Advances in Applied Agricultural Sciences 2 (2014); 11: 58-69
Seed priming with aqueous plant extracts improved seed germination and seedling growth under chilling stress in Lentil (Lens culinaris Medik)
Shakeel Imran 1*, Irfan Afzal 1, Muhammad Amjad 2, Ahsan Akram 3,Khalid Mahmood Khawar 4 and Seef Pretorius 5
1 Department of Crop Physiology, University of Agriculture, Faisalabad, 38040, Pakistan.
2 Department of Soil Science and Soil & Water Conservation, PMAS-Arid Agriculture University, Rawalpindi, Pakistan.
3 Institute of Horticultural Sciences, University of Agriculture, Faisalabad, 38040, Pakistan.
4 Department of Field Crops, University of Ankara, 06110 Diskapi-Altindag, Ankara, Turkey.
5 Department of Soil, Crop and Climate Sciences; University of the Free State, South Africa.
Application of commercial antioxidants, vitamins or nutrients for seed enhancements is very expensive for resource poor farmers. This study was planned to inspect the potential of Aloe vera, Moringa olifera or sugar beet aqueous extracts for germination and seedling vigor enhancement in lentil under controlled conditions (10±1°C and 25±1°C). Lentil seeds were soaked in aerated solutions of A. vera leaf extract (ALE), M. olifera leaf extract (MLE) and sugar beet root extract (SRE) for 14 h using five concentrations (1%, 2%, 3%, 4% and 5%) of each. Water soaked and untreated seeds were used as control. Priming with 2% ALE, 3% MLE and 2% SRE were the most effective in boosting up germination rate and succeeding seedling growth under chilling conditions. Better performance of seedlings was the consequence of decreased time to 50% germination (T50) and mean germination time (MGT); elevated germination index (GI) and final germination percentage (FGP). Improved seedling growth with ALE and SRE or MLE was a feature of increased shoot, root lengths and enhanced seedling weights compared with control. Enhanced performance of lentil seedlings with priming of 2% ALE, 3% MLE and 2% SRE was an attribute of superior α-amylase activity and sugar contents under both temperature limits.
Lentil (Lens culinaris Medik.) is largely grown in South East Asia and generally consumed as chunky soup made from whole grain or split pulse (Zia-ul-Haq et al., 2011). Lentils are excellent source of protein and also rich in important vitamins, minerals, soluble and insoluble dietary fibre, so often termed as ‘poor man’s meat’ (Bhatty, 1988). After chickpea, it is the second largest rain fed grown winter legume (Ayub et al., 2001). There is short time water availability under arid and semiarid environments so successful crop establishment depends on early, rapid and uniform seed germination under stressful conditions (Windauer et al., 2007). Nonetheless, chances for achieving a good crop yield would be high if the stress effect can be lessened at the germination stage (Afzal et al., 2012).
Lentil is usually gown in mid-winter when the soil temperature is very low for optimum germination. Seeds sown when, the soil temperature is 10°C or lower often injured by cold water imbibitions (Cohn and Obendorf, 1978). Sometimes, soil temperature goes below 0°C during night time in south Asia, causing delay in germination and reduced seedling emergence (Basra et al., 2011). Moreover, low temperature may induce chilling injury through production of reactive oxygen species which cause oxidative damage to various macromolecules and cellular structures (Noctor and Foyor, 1998) and limit essential plant nutrients especially potassium (Carry and Berry, 1978).
Synchronised germination and improved crop stand at suboptimal temperatures can be achieved through seed priming (Basra et al., 1988; Afzal et al., 2008). Integration of plant growth regulators, vitamins or nutrients during seed priming resulted in enhanced seed performance and early plant growth and development, particularly under adverse conditions, such as temperature extremes or salinity (Bakht et al., 2011). However, resource poor farmers cannot use expensive plant hormones, antioxidants or nutrients for seed priming (Basra et al., 2011; Imran et al., 2013). So, there is a need to explore natural and environment friendly plant growth enhancers which should be reliable and economical under prevailing circumstances. So, this study was planned to investigate the potential of Aloe vera, Moringa olifera and sugar beet aqueous extracts as seed priming agents.
A. vera has been used for several centuries for its healing and curative properties while over 75 active ingredients from its inner gel have been identified (Habeeb et al., 2007). A. vera leaf gel is a very excellent source of plant nutrients- calcium, iron, magnesium, potassium, phosphorous and zinc (Dagne et al., 2000); enzymes- amylase, catalase, lipase, oxidase and superoxide dismutase (Vazquez et al., 1996); amino acids- Alanine, glycine, leucine and proline (Reynolds and Dweck, 1999); Vitamins- B complex, C, β-carotene and α-tocopherol (Vinson et al., 2005) and other organic compounds- triglicerides, triterpenoid, gibberillin, potassium sorbate and salicylic acid (Hamman, 2008).
Moringa leaves are very good source of zeatin, cytokinin, potassium, calcium, protein, ascorbate, vitamin A and C (Foidl et al., 2001). Vitamin A and other micronutrient deficiencies can be overcome by Moringa application (Nambiar, 2006). Use of MLE as a seed priming agent has been found to improve germination and seedling vigour of maize under optimal (Basra et al., 2011) and stressful condition (Afzal et al., 2012; Imran et al., 2013), however the effects under low temperature stress conditions on legumes have not been judged earlier.
Sugar beet (Beta vulgaris) is rich in sugars and glycine betaine (GB) was first discovered in its juice (Mack et al., 2007). GB increases the water retention of plant cells by protecting from osmotic inactivation (Makela, 2004). Physiologically, it produces methyl groups to facilitate various biochemical processes and acts as an osmolyte to protect cells from abiotic stresses (Craig, 2004). Moreover, exogenous application of SRE is superior to GB and it can be used as a substitute cheaper source of GB for protecting plants against the destructive effects of salinity (Abbas et. al., 2010).
Thus, we hypothesised that application of these natural growth enhancers and ameliorative agents can escape the lentil seeds from chilling stress by providing uniform early germination with better seedling establishment.
Materials and Methods
Lentil seeds cv. NIAB Masoor-2006 (93% germination) were procured from Nuclear Institute of Agriculture and Biology, Faisalabad, Pakistan. The initial moisture content was 13.6%. Seeds were surface sterilised in 1% sodium hypochlorite solution for 3 minutes and then rinsed with sterilised water and air-dried.
Fresh mature A. vera and moringa leaves were obtained from Medicinal Plant Nursery, University of Agriculture, Faisalabad. Healthy sugar beet roots were purchased from local vegetable market. All items were washed thoroughly and chilled overnight before extraction. Extraction was done with locally assembled machine and extracts were sieved and stored at -20oC for further use.
For experimentation, 1%, 2%, 3%, 4% and 5% diluted aqueous extracts of each were prepared and seeds were soaked in respective aerated solutions for 14 h at room temperature. Untreated and water soaked seeds were taken as control. After each treatment, seeds were rinsed thoroughly with distilled water and dried back closer to original moisture level under shade condition, sealed in polythene bags and stored in a refrigerator at 5°C until use (Lee et al., 1998).
Primed and non-primed seeds were sown on two moistened layers of filter paper in an incubator (Sanyo, England) constantly at 25±1°C and 10±1°C for one week. Twenty five seeds were placed in each Petri dish with five replications and considered germinated on radicle visibility. Germination was counted on daily basis. Seedlings were harvested and observations were taken regarding seedling growth according to ISTA protocols (ISTA, 2010).
Time taken to 50% germination (T50) was calculated according to the following formula of Coolbear et. al.,1984.
Where N is the number of final emergence count and ni, cumulative number of seeds emerged at adjacent days ti and tj when ni < (N+1)/2 < nj.
Mean emergence time (MET) was calculated according to following equation of Ellis and Roberts, 1981.
Where n is the number of seeds germinated on day D, and D is the number of days calculated from the beginning of emergence.
Germination index (EI) was calculated using following formula, Association of Official Seed Analysts (AOSA, 1983).
For computing total soluble sugars, 1 g of ground seed was hydrolyzed with 2.5 N HCl for 3 h in a boiling water bath.
Following cooling, the liquid was neutralized with Na2CO3 until effervescence ended; the volume was made up to 100 ml with distilled water then centrifuged at 10,000 xg for 10 min at 4oC and the supernatant was collected. Total soluble sugars were determined by method suggested by Hedge and Hofreiter (1962) and Thimmaiah (2004) with rapid and convenient anthrone reagent. In the same way, reducing sugars were measured by DNS method reported by Miller (1959) and Sadasivam and Manickam (1992) from the seed sample (1 g) extracted in 80% ethanol twice (5 ml each time).
α-amylase activity was found out after extraction of lentil seed (2 g) in potassium phosphate buffer (pH: 7.0) while phenyl methyl sulfonyl fluoride (PMSF) (10 mM) was added as proteases inhibitor. The samples were centrifuged for 10 minutes at 10000 xg at 4°C and the supernatants were used for calculation of the α-amylase activity by the modified DNS method (Varavinit et al., 2002).
Experiments were repeated twice with five replicates. Data were analyzed with SPSS statistical package while bar graphs were computed through Micro soft excel computer program.
Germination and Seedling Vigor Evaluation at 25oC
All the priming treatments significantly improved FGP and GI as compare to untreated and HP seeds (Table 1). High FGP and GI are the signs of improved and synchronized germination. Maximum FGP was observed in 2% SRE, 3% MLE and 2% ALE followed by 2% MLE, 4% MLE, 5% MLE, 3% ALE, 4% ALE and 3% SRE whereas minimum was recorded in control and HP. Almost similar trend was examined in case of GI. Highest GI was examined in 3% MLE, 2% SRE, 2% ALE and 4% MEL chased by 3% SRE, 3% ALE, 2% MLE and 4% SRE while lowest was detected in control trailed by HP, 1% ALE, 1% SRE and 1% MLE. Other concentrations showed intermediate effect. Low T50 and MGT are the indices of better germination. The smallest T50 and MGT were observed in 3% MLE, 2% SRE, 2% ALE and 2% MLE proceeded by 3% ALE, 5% MLE, 3% SRE and 4% ALE whilst biggest value was achieved in control and HP followed by 5% ALE, 1% ALE, 1% MLE and 5% SRE.
Increased shoot length was recorded in 2% SRE, 3% MLE, 3% SRE, and 4% SRE pursued by 5% ALE, 3% ALE, and 1% ALE while decreased was observed in control and HP. Maximum root length was linked with 3% MLE, 2% ALE, 1% ALE, 2% SRE, 3% ALE and HP chased by 5% ALE, 3% SRE, 4% SRE whereas minimum was attached with control. Highest seedling fresh and dry weights were attributed with 2%, SRE, 3% SRE, 1% SRE, 3% MLE, 2% ALE, and 1% ALE ensue by 5% SRE, 4% ALE and HP where lowest was found in control. Elevated root and shoot ratio was inspected in 2% ALE, HP, 4% ALE, and control as well. Remaining treatments gave mixed response to ratio of root and shoot.
Germination and Seedling Vigor Evaluation at 10oC
Seed priming with natural growth promoters resulted in improved germination and seedling vigor even at low temperature.
Table 1. Effect of seed priming treatments on germination potential and seedling growth oflentil under optimum conditions (250C)
Shoot L. (cm)
Means showing different letters are significantly different at 5% probability level. HP= Hydropriming, ALE= A. vera leaf extract, MLE= M. olifera leaf extract, SRE= Sugar beet root extract, FGP= Final germination percentage, T50= Time to 50% germination, GI= Germination index, MGT= Mean germination time, SLFW= Seedling fresh weight, SLDW= Seedling dry weight
Table 2. Effect of seed priming treatments on germination potential and seedling growth oflentil under cool conditions (100C)
Shoot L. (cm)
Means showing different letters are significantly different at 5% probability level. HP= Hydropriming, ALE= A. vera leaf extract, MLE= M. olifera leaf extract, SRE= Sugar beet root extract, FGP= Final germination percentage. T50= Time to 50% germination, GI= Germination index, MGT= Mean germination time, SLFW= Seedling fresh weight, SLDW= Seedling dry weight
Fig. 1. Effect of A. vera leaf extract (ALE) priming treatments on biochemical attributes of lentil during germination assay. Where, C= Control and HP= Hydropriming.
Maximum FGP was recorded in 3% MLE, 2% SRE, 2% ALE, 4% MLE and 5% MLE followed by 3% ALE, 3% SRE and 1% MLE while minimum was observed in control and HP. Other treatments resulted in mixed response. Lower values of T50 were attached with 3% MLE, 2% SRE, 2% ALE, 4% MLE and 2% MLE followed by 3% ALE, 4% SRE, 5% MLE and 3% SRE whilst higher were linked with control and HP. Elevated GI was examined in 3% MLE, 2% SRE, 2% ALE and 2% MLE trailed by 3% SRE, 3% ALE, 1% SRE and 4% MLE whereas minimum was found in control and HP. Minimum MGT was inspected in 3% MLE, 2% SRE, 2% ALE, 3% SRE and 4% MLE followed by 2% MLE, 1% MLE, 3% ALE and 4% SRE where maximum was investigated in control and HP. Leftover treatments showed midway response.
Seedling vigor was also significantly enhanced at cool conditions. Highest shoot and root lengths were found in 4% ALE, 5% ALE, 3% MLE, 2% SRE, and 4% SRE chased by 3% ALE, 3% SRE, 4% MLE, 2% MLE and 1% SRE where lowest was achieved in control and HP. Maximum seedling fresh and dry weight was observed in 2% ALE, 4% ALE, 1% ALE, 2% SRE, 4% MLE and 1% SRE followed by 3% SRE, 3% SRE, 5% MLE and 3% ALE while minimum was recorded in 2% MLE, control and HP ensued by 1% MLE and 3% ALE. Higher ratio of root and shoot was attributed with HP, control 2% ALE, 2% SRE and 1% ALE but lower was linked featured in 5% ALE, 5% MLE and 4% MLE. Lingering treatments gave intermediary effect on seedling vigor.
On the other hand primed and unprimed seeds were analyzed to judge the sugars (total and reducing) and α-amylase activity. In case of ALE priming (Figure 1), maximum total sugars were computed in 3% and 2% way maximum α-amylase activity found in 2% ALE followed by 1% and 4% ALE while higher reducing sugars were recorded in 2% and 3% ALE. In the same and 3% ALE followed by 1% and 4% ALE. The lowest biochemical activity of sugars and α-amylase was recorded in control and HP.
Fig. 2. Effect of M. olifera leaf extract (MLE) priming treatments on biochemical attributes of lentil during germination assay. Where, C= Control and HP= Hydropriming.
Moreover, in SRE priming (Figure 3); maximum total sugars were recorded in 2% and 3% SRE trailed by 1% and 4% SRE whereas higher reducing sugars were computed in 2% and 3% SRE followed by 4% and 5% SRE. Likewise, maximum α-amylase activity recovered in 3% and 4% ALE chased by 2% and 4% SRE. The lowest biochemical activity of sugars and α-amylase was found in control and HP.
This study was planned according to the need of the era with special focus on resource deprived farmers attached with traditional crop production practices. Priming with natural growth promoters proved effective at both growing conditions. All priming treatments significantly improved FGP and GI keeping T50 and MGT lower as compare to control and HP seeds. Seed priming can effectively be used to improve germination and seedling establishment under low temperature conditions (Afzal et al., 2008).
Germination and seedling attributes were significantly improved by all priming concentrations as compare to control and HP seeds (Table 1). Though a marked reduction in germination and seedling vigor was observed under cool conditions (Table 2) but seed priming with 2% ALE, 3% MLE and 2% SRE successfully mitigated the adverse effects of chilling stress. It was might be due to presence of natural antioxidents, mineral nutrients, osmoprotectants and plant growth hormones. Similar finding have earlier been reported by (Basra et al., 2011; Afzal et al., 2012; Imran et al., 2013).
Under low temperature conditions, MLE performed better in improving germination speed and final germination count while SRE was successful in providing maximum seedling biomass whereas ALE showed a mixed response by providing better germination and seedling growth under both conditions. This was might be due to integration of growth enhancers during seed priming/soaking period resulting in early activation of germination regulators as compare to control. Analogous findings have former been presented by Mahmood et al., 2009 and Nouman et al., 2012. MLE is a good source of cytokinin which promotes early germination by enhancing cell division (Basra et al., 2011) while SRE is rich in GB which is responsible for stomatal regulation by means of osmatic adjustment (Nawaz and Ashraf, 2007) and increase cell turgidity (Genard et al., 1991). Chilling stress provokes generation of ROS, which may react with important macromolecules causing oxidative injury resulting in spoiling the optimal cellular functions (Farooq et al., 2008).
As earlier mentioned, ALE is loaded with a variety of plant hormones, vitamins and nutrients. The improved performance of ALE was might be due to the increased contents of ascorbate (Afzal et al., 2007), proline (Shakirova et al., 2003) or H2O2 signaling effect that enhances the tissue K+, NO3– and PO43-levels (Wahid et al., 2007).
Fig. 3. Effect of Sugar beet root extract (SRE) priming treatments on biochemical attributes of lentil during germination assay. Where, C= Control and HP= Hydropriming
Higher root and shoot lengths coupled with increased seedling fresh and dry weights are the attributes of early and rapid germination resulting in higher seedling vigor. An increase in sugar contents was found in most promising priming concentrations as compare to untreated or HP seeds that are correlated with superior seed vigor and findings verify the verdicts of Horii et al., 2007 and Afzal et al., 2012.
In conclusion, seed priming was successful in inducing chilling tolerance in lentil seeds through increased vigour associated with carbohydrate metabolism and hydrolytic enzyme activities. Priming with 2% ALE, 3% MLE or 2% SRE was found most effective and can be suggested to farmers for achieving higher yields under cool conditions.
Abbas, W., M. Ashraf and N. A. Akrama. (2010). Alleviation of salt-induced adverse effects in eggplant (Solanum melongena L.) by glycinebetaine and sugarbeet extracts. Scientia Horticulturae, 125: 188–195.
Afzal, I., S.M.A. Basra, N. Ahmad and M. Farooq. (2007). Optimization of hormonal priming techniques for alleviation of salinity stress in wheat (Triticum aestivum L.). Caderno de Pesquisa Serie Biologia, 17: 95-109.
Afzal, I., S.M.A. Basra, M. Shahid and M. Saleem. (2008). Priming enhances germination of spring maize in cool conditions. Seed Science and Technology, 36: 497-503.
Afzal, I., Hussain, B., S.M.A. Basra and Hafeez, R. (2012). Priming with moringa leaf extract reduces imbibitional chilling injury in spring maize. Seed Science and Technology, 40: 271-276.
AOSA (1983). Seed Vigor Testing Handbook. Contribution No. 32 to the Handbook on Seed Testing. Association of Official Seed Analysts. Springfield, IL.
Ayub K, M. Rahim and A. Khan. (2001). Performance of exotic lentil varieties under rainfed conditions in Mingora (NWFP) Pakistan. Journal of Biological Sciences, 1: 343-344.
Bakht, J., Shafi, M., Jamal, Y. and Sher, H. (2011). Response of maize (Zea mays L.) to seed priming with NaCl and salinity stress. Spanish Journal of Agricultural Research, 9: 252-261.
Basra, S.M.A.., Bedi, S. and Malik, C.P. (1988). Accelerated germination of maize seeds under chilling stress by osmotic priming and associated changes in embryo phospholipids. Annals of Botany, 61: 635-639.
Basra, S.M.A., Iftikhar, M.N. and Afzal, I. (2011). Potential of moringa (Moringa oleifera) leaf extract as priming agent for hybrid maize seeds. International Journal of Agriculture and Biology, 13: 1006-1010.
Bhatty, R.S. (1988). Composition and quality of lentil (Lens culinaris Medik): a review. Canadian Institute of Food Science and Technology Journal, 21: 144-160.
Carry, R.W. and J.A. Berry. (1978). Effects of low temperature on respiration and uptake of rubidium ions by excised barely and corn roots. Plant Physiology, 61: 858-860.
Cohn, M.A. and R.L. Obendorf. (1978). Occurrence of stellar lesion during imbibitional chilling of Zea mays L. American Journal of Botany, 65: 50-56.
Coolbear, P. Grierson, D. and Heydecker, W. (1980). Osmotic pre-sowing treatments and nucleic acid accumulation in tomato seeds (Lycopersicon lycoperciscum). Seed Science and Technology, 8: 289-303.
Craig, S.A., (2004). Betaine in human nutrition. American Journal of Clinical Nutrition, 80: 539–549.
Dagne, E.; Bisrat, D.; Viljoen, A.; Van Wyk, B-E. (2000). Chemistry of Aloe species. Current Organic Chemistry, 4: 1055-1078.
Ellis, R.A. and Roberts, E.H. (1981). The quantification of ageing and survival in orthodox seeds. Seed Science and Technology, 9: 373-409.
Farooq, M., T. Aziz, S.M.A. Basra, M.A. Cheema and H. Rehman. (2008). Chilling tolerance in hybrid maize induced by seed priming with salicylic acid. Journal of Agronomy and Crop Science, 194: 161-168.
Genard, H., Saos, J.L., Hillad, J., Tremolieres, A., Boucaud, J. (1991). Effect of salinity on lipid composition, glycinebetaine content and photosynthetic activity in chloroplasts of Suaeda maitima. Plant Physiology and Biochemistry, 29: 421–427.
Habeeb, F.; Shakir, E.; Bradbury, F.; Cameron, P.; Taravati, M.R.; Drummond, A.J.; Gray, A.I.; Ferro, V.A. (2007). Screening methods used to determine the anti-microbial properties of Aloe vera inner gel. Methods, 42: 315-320.
Hamman, J. H., (2008). Composition and Applications of Aloe vera Leaf Gel. Molecules, 13: 1599-1616.
Hedge, J.E. and Hofreiter, B.T. (1962). Carbohydrates Chemistry. Ed. 17. Academic Press, New York.
Horii, A., McCue, P. and Shetty K. (2007). Seed vigour studies in corn, soybean and tomato in response to fish protein hydrolysates and consequences on phenolic-linked responses. Bio resource Technology, 98: 2170-2177.
Imran, S., I. Afzal and S.M.A. Basra and M. Saqib, (2013). Integrated seed priming with growth promoting substances enhances germination and seedling vigour of spring maize at low temperature. International Journal of Agriculture and Biology, 15: 1251‒1257.
ISTA. (2010). International Rules for Seed Testing. ISTA Secretariat, Switzerland.
Thimmaiah, S.R. (2004). Standard methods of Biochemical Analysis. Kalyani Publishers, New Dehli, India, pp. 54-55.
Lee, S.S., J.H. Kim, S.B. Hong and S.H. Yun. (1998). Effect of humification and hardeninig treatment on seed germination of rice. Korean Journal of Crop Science, 43: 157-160.
Mack, G., Hoffmann, C.M., Marlander, B. (2007). Nitrogen compounds in organs of two sugar beet genotypes (Beta vulgaris L.) during the season. Field Crop Research, 102: 210–218.
Mahmood, T., Ashraf, M., and Shahbaz, M. (2009). Does exogenous application of glycinebetaine as a pre-sowing seed treatment improve growth and regulate some key physiological attributes in wheat plants grown under water deficit conditions? Pakistan Journal of Botany, 41: 1291–1302.
Miller, G.I. (1959). Use of dinitrosalicylic acid reagent for the determination of reducing sugars. Annals of Chemistry, 31: 426-428.
Nambiar, V. S. (2006) Nutritional potential of drumstick leaves: an Indian perspective. In: Moringa and other Highly Nutritious Plant Resources: Strategies, Standards and Markets for a Better Impact on Nutrition in Africa. Accra, Ghana.
Nawaz, K. and Ashraf, M. (2007). Improvement in salt tolerance of maize by exogenous application of glycinebetaine: growth and water relations. Pakistan Journal of Botany, 39: 1647–1653.
Noctor, G. and C.H. Foyor. (1998). Ascorbate and glutathione: keeping active Oxygen under control. Annual Review of Plant Physiology and Molecular Biology, 49: 249-279.
Nouman W, Siddiqui MT, Basra S.M.A. (2012) Moringa oleifera leaf extract: An innovative priming tool for rangeland grasses. Turkish Journal of Agriculture and Forestry, 36: 65-75.
Reynolds, T.; Dweck, A.C. (1999). Aloe vera leaf gel: a review update. Journal of Ethnopharmacology, 68: 3-37.
Sadasivam, S. and Manickam, A. (1992). Biochemical Methods for Agricultural Sciences, Wiley Eastern Limited, New Dehli, pp. 11-12.
Shakirova, F.M., A.R. Sakhabutdinova, M.V. Bezrukova, R.A. Fathkutdinova and D.R. Fatkhutdinova. (2003). Changes in the hormonal status of wheat seedlings induced by salicylic acid and salinity. Plant Science, 164: 317-324.
Varavinit, S. Chaokasem, N. and Shobsngob, S. (2002). Immobilization of a thermostable α-amylase. Science Asia, 28: 247-251.
Vazquez, B.; Avila, G.; Segura, D.; Escalante, B. (1996). Antiinflammatory activity of extracts from Aloe vera gel. Journal of Ethnopharmacology, 55: 69-75.
Vinson, J.A.; Al Kharrat, H.; Andreoli, L. (2005). Effect of Aloe vera preparations on the human bioavailability of vitamins C and E. Phytomedicine, 12: 760-765.
Wahid, A. (2007). Physiological implications of metabolite biosynthesis for net assimilation and heat stress tolerance of sugarcane (Saccharum officinarum) sprouts. Journal of Plant Research, 120: 219–228.
Windauer, L., Altuna, A. and Benech-Arnold, R. (2007). Hydrotime analysis of Lesquerella fendleri seed germination responses to priming treatments. Industrial Crop Production, 25: 70–74.
Zia-ul-haq, M., S, Ahmad., M, Aslam S., S, Iqbal., M, Qayum., A, Ahmad., D. L. Luthria and R, Amarowicz. (2011). Compositional studies of lentil (Lens culinaris medik.) cultivars commonly grown in Pakistan. Pakistan Journal of Botany, 43: 1563-1567.