Impaginato 227 Adv. Hort. Sci., 2019 33(2): 227-233 DOI: 10.13128/ahs-23812 Foliar application of asparagine and casein on biochemical and morphological attributes of garden cress (Lepidium sativum L.) under greenhouse conditions A. Jorkesh 1 (*) , M.H. Aminifard 2 1 Department of Horticulture, Faculty of Agriculture, University of Guilan, Rasht, Iran. 2 Department of Horticulture Science, Special Plants Regional Research Centre, Faculty of Agriculture, University of Birjand, Iran. Key words: Foliar spray, nitrogen content, phosphorus content, vegetative stage. Abstract: In this study, the effect of foliar application of Asparagine (ASN) and Casein (CSN) during vegetative stage at four rates (0, 50, 100 and 150 mg l-1) was investigated on garden cress (Lepidium sativum L.). The results showed that asparagine application, especially at a high level, could significantly increase the morpho-physiological traits such as plant height, leaf and stem fresh weights and leaf and stem dry weights, leaf pigments (chlorophyll a and chlorophyll b) and leaf nutrients content (Nitrogen and Phosphorus). Also, the results indicated that casein application at 50 mg l-1 rate had the best perfor- mance through in stem and root fresh weights, stem dry weight and diameter of main stem traits. Casein application at rate 100 mg l-1 had the highest leaf nitrogen and phosphorus content. Generally, our findings suggest that the use of asparagine and casein can be considered as an appropriate growth regulator in garden cress cultivation. 1. Introduction Garden cress (Lepidium sativum L.) is an edible herb and a member of the Cruciferae (Brassicaceae) family. It is commonly cultivated throughout the temperate regions of India and Pakistan (Nadkarni, 1954). The plant is cultivated as culinary vegetable all over Asia (Nadkarni, 1976). Garden cress is an annual standing plant, growing up to 30 cm. It is a well known cookable herb and the leaves are widely used as a garnish in salads. In addition to its leaves that have medicinal properties, the seeds are aperi- ents, diuretic, tonic, demulcent, aphrodisiac, carminative (Chopra et al., 1986). Moreover, the seeds, which are used in folk therapies, have many activities like thermogenic, depurative, rubefacient, tonic, aphrodisiac, abortive, ophthalmic, diuretic (Gokavi et al., 2004; Dugasani et al., 2009). Intensive farming practices, which produce high yields and quality, require the extensive use of chemical fertilizers that are both costly and (*) Corresponding author: a.jorkesh@gmail.com Citation: JORKESH A., AMINIFARD M.H., 2019 - Foliar appli- cation of asparagine and casein on biochemical and morphological attributes of garden cress (Lepidium sativum L.) under greenhouse condi- tions. - Adv. Hort. Sci., 33(2): 227-233 Copyright: © 2019 Jorkesh A., Aminifard M.H. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 10 August 2018 Accepted for publication 18 February 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(2): 227-233 228 create environmental problems. Therefore, there has been a recent resurgence of interest in environmen- tally friendly, sustainable and organic agricultural practice (Orhan et al., 2006). Thus, it is necessary to supply the plant requirement to nutrient through proper procedure. There are different ways to supply plant nutrient’s requirement such as soil feeding and foliar application. Of these, foliar feeding is an effec- tive method for improving soil deficiencies and over- coming the soils inability to transfer nutrients to the plant. It has reported the foliar feeding can be 8 to 10 times more effective than soil feeding and up to 90% of a foliar fed nutrient solution can be found in the smallest root of a plant within 60 minutes of applica- tion (Garcia and Hanway, 1976). Amino acids are the major building element for proteins (Andrews, 1986). Amino acids is a well- known biostimulant which has positive influence on plant growth, yield and significantly decrease the damages caused by abiotic stresses (Kowalczyk and Zielony, 2008). ASN is widely used as a source of organic nitrogen in the media upon which certain bacteria are grown (Long and Seibert, 1926). ASN is thought to play a clearly important role in the trans- portation and storage of nitrogen (Lehmann and Ratajczak, 2008), because of their relatively stable nature and high N⁄C ratio (Ireland and Lea, 1999; Masclaux-Daubresse et al., 2006). In plants, ASN, one of the most prevalent amides, has been reported to be the primal source of Nitrogen for protein synthe- s i s , p a r t i c u l a r l y i n a c t i v e l y g r o w i n g t i s s u e s (Brouquisse et al., 1992). ASN aggregation in plants in response to environmental stress could be an ammo- nium detoxification mechanism and a means to stock up Nitrogen when protein synthesis is impaired in plants due to stressful environments (Herrera- Rodríguez et al., 2007). CSN is a very rich source of essential amino acids (Sarode et al., 2016). There are four different CSN proteins (αs1, αs2, β and K), which are different in their amino acid composition (Dalgleish, 1989). Among proteins, CSN has been vastly used in artificial rearing diets because it contains all the essential amino acids, is soluble in water, and does not coagu- late or precipitate after heating (Parra, 1979). CSN also contains important substances such as fatty acids, cholesterol, sugars, vitamins, and minerals (Vanderzant, 1974). CSN contains 0.7-0.9% phospho- rus. So, CSN is known as a phosphoprotein (Sarode et al., 2016). Several pre-harvest factors like climatic conditions and available nutrients can influence on yield and quality of vegetables (Lee and Kader, 2000). In spite of wide rang of properties, there are a few study about the effect of amino acids on medicinal plants. So this study was conducted to evaluate the effects of ASN and CSN foliar application on vegeta- tive and reproductive growth, physiological and bio- chemical traits of garden cress. 2. Materials and Methods In order to investigate the response of Lepidium sativum to foliar application of asparagine (ASN) and casein (CSN) two separate pot experiments were conducted at research greenhouse of University of Guilan, Iran. The experiments were carried out based on two randomized complete design with three repli- cations. The treatments in this study were different levels of ASN and CSN (0, 50, 100 and 150 mg l-1). Foliar application of experimental solutions was started from the four-leaf stage and done once every two weeks in the morning, up to the early May. In each spraying date, 20-25 ml of corresponding solu- tion was applied per pot. Garden cress seeds which were prepared from Isfahan-PakanBazr Company were planted in 6th January. The sowing was done in pots with the 33 cm diameter and 22 cm height. The seed bed was a mixture of soil, cow manure and sand with the ratio of 2:1:1. The main properties of soil used in the pots are shown in Table 1. Except for cow manure, no fertilizer was used during the plants growth cycle. Plants were irrigated every 10 days. Plant thinning was performed 15 days after emer- gence, so that, 6 plants were kept in each pot. During plant growth the greenhouse temperature was 25˚C on the day and 15°C during the nights. In addition, CO2 concentration was 350 ppm, relative humidity was 40% and photoperiod adjusted as 16 hours’ light and 8 hours of darkness. Organic carbon (%) Nitrogen (%) Calcium (%) Phosphorous (%) Potassium (%) pH Texture 1.2 1 0.48 0.19 0.55 7.1 Loam sandy Table 1 - Some chemical indices of soil used in pots for garden cress cultivation Jorkesh and Aminifard - Foliar application on Lepidium sativum under greenhouse conditions 229 In 4th May, four plants were lifted form the soil in each pot for determination of some above- and below-ground vegetative indices. The measured indices were plant height, leaf weight, root weight, stem weight and main stem diameter (by a ruler). The remained two plants were used to measure some reproductive traits including number of florets. The pigments content (carotenoid, chlorophyll a and chlorophylls b) was measured by the method has been explained by Minguez-Mosquera and Prez- Galvez (1998). To determine the leaf nitrogen, phos- phorous and calcium percentages, the method of Jones (2001) was used. The method of Bakhshi and Arakava (2006) also was used for the extraction of leaf extract to deter- mine phenolic compounds and antioxidant capacity. Then, phenols were analyzed by using the method of Folin-Ciocalteau reported by Tavarini et al. (2008). For this purpose, the absorbance at 760 nm was measured using a spectrophotometer (T80+PG Instrument UV/Vis Spectrometer) and the values were expressed as mg gallic acid/100 g fresh weight. Antioxidant capacity was determined using DPPH free radical scavenging method which has been described by Sanchez-Moreno et al. (1999). Finally, data analysis was done using SAS 9.2 software and means were separated via Tukey’s (honest significant difference) test at the 5% level of probability. 3. Results and Discussion Vegetative and reproductive growth The analysis of variance of the investigated mor- phological traits was presented in Table 2. The results revealed that both ASN and CSN significantly affected the plant length, leaf and stem fresh weight, leaf and stem dry weight. So that, the greatest plant height w a s o b s e r v e d a t a r a t e o f 1 5 0 m g l - 1 o f A S N . Moreover, applying ASN at rate of 100 mg l-1 had the highest leaf and stem fresh weights and leaf and stem dry weights. Casein application significantly affected the stem and root fresh weights, stem dry weight and diame- ter of main stem (Table 2). So that, the plants grown under CSN spray at rate of 50 mg l-1 had the best per- formance due to stem dry and fresh weight, root fresh weight and main stem’s diameter. Asparagine and CSN foliar spraying could improve the morphological characteristics in garden cress. These results are in agreement with the previously studies by Kaya et al. (2013) on maize, Rasmia et al. (2014) on palm, Saeed et al. (2005) on soybean, Akladious and Abbas (2013) and El-Desouky et al. (2011) on tomato, Shafeek and Helmy (2012) on onion. Also, El-Zohiri and Asfour (2009) on potato found that spraying of amino acids at 0.25 ml/L sig- nificantly increased vegetative growth expressed as plant height and dry weight of plant. Their finding indicated that amino acids could improve the vegeta- tive and reproductive traits in plants. These ability my be due to their important role in plant metabo- lism and protein assimilation which is necessary for cell formation and consequently increase the fresh and dry matter (Fawzy et al., 2012). Amino acids contribute to the synthesis of growth hormones; therefore, it can be concluded that an increase in cell division and cell enlargement is the r e a s o n b e h i n d e n h a n c e d g r o w t h p a r a m e t e r s (Shafeek and Helmy, 2012). The positive effect of amino acids on growth was stated by Goss (1973) who indicated that amino acids can serve as a source Table 2 - Mean squares for the effect of different levels of Aspargine (ASN) and Casein (CSN) on vegetative and reproductive indices of garden cress Traits Source of Variation Treatment Error CV ASN CSN ASN CSN ASN CSN Plant height 24.22 * 5.86 NS 3.75 10.41 5.75 10.38 Leaf fresh weight 0.66 * 1.008 NS 0.09 0.25 5.09 7.97 Leaf dry weight 0.27 ** 0.04 NS 0.03 0.02 11.69 12.28 Root fresh weight 0.17 NS 0.2 ** 0.06 2 8.52 5.22 Root dry weight 0.002 NS 0.004 NS 0.01 0.005 8.54 5.94 Stem fresh weight 2.65 ** 2.001 ** 0.04 0.16 3.62 6.83 Stem dry weight 0.5 ** 0.22 * 0.02 0.04 9.66 14.13 Diameter of main stem 0.04 NS 0.11 * 0.05 0.01 10.98 5.87 Number of florets 0.22 NS 0.75 NS 0.66 0.5 11.66 10.47 NS, *, ** Non-significant, significant at 5%, and 1% probability level, respectively. Adv. Hort. Sci., 2019 33(2): 227-233 230 of carbon and energy when carbohydrates become deficient in the plant’s releasing the ammonia and organic acid form which the amino acid was originally formed (Table 3). The organic acids then enter Kerb’s cycle, to be broken down to release energy through respiration (Goss, 1973). Serna et al. (2012) found that the spray of pepper plants with a mixture of amino acids led to raise the efficiency of photosyn- thesis, and thus, give the best vegetative growth. Biochemical traits and nutrients content The mean squares of physiological and mineral content are presented in Table 4. Total phenolic compounds and antioxidants activity were not affect- ed by foliar spraying of ASN and CSN (Table 5). Chlorophyll a and b content significantly increased in response to foliar ASN treatments (Table 4). The highest contents of chlorophyll a and b observed in plants grown under spraying of ASN at rate of 100 Table 3 - Means comparison for the effect of different levels of Asparagine and Casein on some vegetative and reproductive growth parameters of garden cress Means with the same letter(s) within a column are not significantly different (P≤0.05) based on Tukey's test. Table 4 - Mean squares for the effect of different levels of Aspargine (ASN) and Casein (CSN) on leaf nutrient and pigments content and some biochemical parameters in garden cress NS, *, ** Non-significant, significant at 5%, and 1% probability level, respectively. Traits Source of variation Treatment Error CV ASN CSN ASN CSN ASN CSN Chlorophyll a 0.13 ** 31.66 NS 0.006 0.008 4.9 6.75 Chlorophyll b 0.05 ** 0.01 * 0.001 0.002 5.83 7.89 Carotenoid Content 0.0004 NS 0.003 NS 0.001 0.002 8.32 12.96 Leaf Nitrogen Content 0.09 ** 0.18 ** 0.006 0.01 4.79 6.72 Leaf Phosphorous Content 0.01 * 0.03 ** 0.003 0.002 8.86 6.66 Leaf Calcium Content 0.007 NS 0.0007 NS 0.007 0.003 9.45 6.95 Total Antioxidants 21.41 NS 6.97 NS 12.75 6.16 6.64 4.7 Total Phenol 31.66 NS 6.97 NS 7.33 17.33 4.99 7.81 Treatments (mg l-1) Plant height (cm) Leaf fresh weight (g plant-1) Leaf dry weight (g plant-1) Root fresh weight (g plant-1) Root dry weight (g plant-1) Stem fresh weight (g plant-1) Stem dry weight (g plant-1) Diameter of main stem (mm) Number of florets Aspargine 0 29.66 b 5.47 b 1.18 b 2.87 a 1.20 a 4.77 c 1.15 c 1.99 a 7.00 a 50 34.66 ab 6.05 ab 1.81 a 2.97 a 1.25 a 5.73 b 1.44 bc 2.08 a 6.66 a 100 34.00 ab 6.57 a 1.83 a 3.42 a 1.26 a 6.96 a 2.06 a 2.25 a 7.00 a 150 36.33 a 6.31 a 1.62 ab 3.12 a 1.26 a 6.38 ab 1.86 ab 2.02 a 7.33 a Casein 0 29.66 a 5.47 a 1.18 a 2.87 b 1.2 a 4.77 b 1.15 b 1.99 b 7.00 a 50 31.00 a 6.75 a 1.45 a 3.39 a 1.22 a 6.64 a 1.80 a 2.37 a 6.33 a 100 30.66 a 6.23 a 1.38 a 2.82 b 1.15 a 6.22 ab 1.58 ab 2.01 b 6.33 a 150 33.00 a 6.65 a 1.43 a 3.14 ab 1.25 a 6.16 ab 1.55 ab 2.3 ab 7.33 a Table 5 - Means comparison for the effect of different levels of Asparagine and Casein on biochemical parameters of garden cress Means with the same letter(s) within a column are not significantly different (P≤0.05) based on Tukey's test. Treatments (mg l-1) Chlorophyll a Chlorophyll b Carotenoid content Leaf nitrogen content Leaf phosphorous content Leaf calcium content Total antioxidants Total phenol Aspargine 0 1.38 b 0.56 c 0.39 a 1.45 b 0.57 b 0.89 a 51.6 a 50.3 a 50 1.53 b 0.73 b 0.4 a 1.6 ab 0.66 ab 0.83 a 53 a 52.6 a 100 1.87 a 0.89 a 0.37 a 1.81 a 0.71 ab 0.95 a 57.6 a 57.3 a 150 1.64 ab 0.71 b 0.35 a 1.81 a 0.73 a 0.87 a 52.6 a 56.3 a Casein 0 1.36 a 0.56 b 0.39 a 1.45 b 0.57 c 0.89 a 51.66 a 50.3 a 50 1.41 a 0.73 a 0.37 a 1.62 ab 0.60 bc 0.86 a 52 a 54.3 a 100 1.45 a 0.70 a 0.44 a 2.01 a 0.78 a 0.82 a 55 a 54.6 a 150 1.38 0.65 ab 0.43 a 1.88 a 0.74 ab 0.87 a 52.3 a 53.6 a Jorkesh and Aminifard - Foliar application on Lepidium sativum under greenhouse conditions 231 needs of the cells are provided with the lowest loss- es. Results of the current study revealed that foliar application of ASN and CSN could increase the growth and yield of garden cress. It could be recom- mended that spraying garden cress plants by ASN (100 and 150 mg l-1) increased the vegetative growth traits (plant height, leaf fresh weight, number of flo- rets), nutrients uptake (leaf nitrogen and phosphorus content) and pigments concentration (chlorophyll a and chlorophyll a) and also application of CSN at 50 mg l-1 level could improved some morphological and biochemical traits of garden cress such as: root fresh weight, stem fresh weight, Chlorophyll b, Leaf nitro- gen and phosphorus content in comparison control treatment. Therefore, it can be concluded that appli- cation of ASN and CSN is a good strategy in garden cress cultivation, but their effectiveness must be also evaluated under environmental stresses, in the future studies. 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