Impaginato 215 Adv. Hort. Sci., 2019 33(2): 215-226 DOI: 10.13128/ahs-23924 The use of organic nano-supplements of fertilizer for lily forcing period A. Hatamzadeh 1, S.-S. Shafiei-Masouleh 2 (*) 1 Department of Horticultural Science, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran. 2 Department of Genetics and Breeding, Ornamental Plants Research Center (OPRC), Horticultural Sciences Research Institute (HSRI), Agricultural Research, Education and Extension Organization (AREEO), Mahallat, Iran. Key words: amylases, chitosan, forcing, flowering, magnetite, magnetic supple- ment of fertilizer, sugars. Abstract: Lilium is one of the most important ornamental plants after roses, carnations and chrysanthemum in the world that is requested as potted of cut flowers. It is so important to consider its quality along with its production rate in terms of yield (quantity). However, it always needs to intend the product costs aside from quantity and quality. Fertilizer utilization is so important and this may be improved by compounds that promote it and also have synergetic effects themselves. We examined both carboxymethylated chitosan (CMC) and magnetic nano-carbocymethylated chitosan (MNCC) to produce Lilium bulb and advised them especially magnetic chitosan. In this study, these compounds (magnetic and non-magnetic chitosan) at concentrations of zero, 2.5, 5, 10 and 15 mg/l were examined during lily forcing in three cultivars, including Cherbourg, Navona, Brunello, which are from Asiatic and Oriental lilies. The results showed that highest concentrations (10 and 15 mg/l) among between examined concentrations regardless of compounds types and cultivars did not make toxicity and had significant effects on plants biology and physiology [con- tents of carbohydrates and enzymes affecting these carbohydrates (amylases)]. However, for observing morphological changes may be need to use higher con- centrations of these compounds. Note that this needs to examine the non-toxi- city of higher concentrations in future studies. 1. Introduction Flowers have always had a valuable place among different classes of society and various ceremonies and rituals. Lilium is one of the most important ornamental plants after roses, carnations and chrysanthemum in the world that is mostly used as cut flowers throughout the year. Furthermore, it is used as potted plants in gardens and green landscapes (Shafiee-Masouleh et al., 2014). It is necessary to consider the nutrition of lilies like other plants in the greenhouse production. And also, optimiza- tion and improvement of photosynthetic efficiency of plants can be effec- tive in increasing photosynthetic storages and thus increasing its quanti- (*) Corresponding author: shafiee.masouleh@areeo.ac.ir Citation: HATAMZADEH A., SHAFIEI-MASOULEH S.-S., 2019 - The use of organic nano-supplements of fertili- zer for lily forcing period. - Adv. Hort. Sci., 33(2): 215-226 Copyright: © 2019 Hatamzadeh A., Shafiei-Masouleh S.-S. 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 14 Septmeber 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): 215-226 216 tative and qualitative yields in terms of flower num- ber, plant size and vase life. Different groups of lilies (Asiatic, Oriental and L. longiflorum) have different nutritional requirements. For example, Asiatic lilies has been reported that have the best growth when are fertilized by lower levels during forcing (Treder, 2003). Chitosan particles in nano-scale can be important in the delivery of drugs in medicine, because these compounds promote the absorption of active mole- cules or compounds through the cell membrane and allow organs to have bioavailability to molecules. Chitosans can be used as encapsulated nanoparticles or used directly and therefore, the surface of these particles with chelating structure of the modified chi- tosan can play an important role in sustainable agri- culture (Kashyap et al., 2015). Magnetic chitosan nanoparticles with the effect of chelating elements in the experiment conducted by Shafiee-Masouleh et al. (2014) increased the biomass of Lilium and the storage organs (bulbs) during bulb production. The effect of magnetic fluids on living systems has been studied both in medicine and in the world of plants, and it has been shown that magnetic fluids can be effective in the production of calluses and metabolic activities. The effects of magnetism on plants by Earth’s magnetic field, pulsed and inductive constant magnetic fields, an electromagnetic field, and effects of magnetic nanoparticles have been studied by researchers and theirs roles have been confirmed to enhance photosynthesis and growth and develop- ment of plants (Pavel et al., 1999; Răcuciu and Creangă, 2007 b; Răcuciu et al., 2009; Shafiee- Masouleh et al., 2014). It has also been shown that superparamagnetic nanoparticles, which are as per- manent magnets effected by external magnetic fields or ambient temperature of their environment, affect membrane systems and membrane ion exchange (Pavel and Creangă, 2005; Faeghi and Seyedpour, 2013). Many researchers investigated the effects of chitin and the chitosan polymers and oligomers as spraying, soil application or seed treatment and fer- tilizer treatments, they reported that photosynthesis and plant growth were enhanced (El-Tantawy, 2009; Dzung et al., 2011; Farouk and Amany, 2012). The allocation of carbon in plants is important that is affected by various factors such as the level of pho- tosynthetic compounds, the number and size of com- peting sink (flowers, seeds, fruits, bulbs and tubers, etc.) and their location in plants and the potential for initial storage in the leaves and re-translocation in plants. Understanding the components that affect the initiation and development of organs and the bal- ance between source and sink organs are essential to regulate the allocation of assimilates (Du Toit, 2001). The chl content of the leaf affects photosynthesis, and Chl a/b is the best index to understand the pho- tosynthetic capacity and direct information on the activity of the enzymes involved in the photosystem II in the chloroplast membrane (Răcuciu and Creangă, 2007 b; Răcuciu et al., 2009). Iron deficiency reduces the amount of photosynthetic pigments. In addition, the electron transfer in the photosystems I and II undergoes a change. Also, the activity of 1, 5-D-phos- phate-carboxylase and the photosynthetic function of plant decrease. Therefore, it is necessary to pro- vide iron for the plant, because the biochemical properties of iron and its effect on the metabolic pathway of the plant are important. Uptaking iron in alkaline soils is difficult; this is due to the formation o f f erri c h yd ro x i d e i n th e p res en ce o f o x ygen , because plant cannot uptake it (Thoiron and Briat, 1999). The main component in reducing NO3- absorp- tion is iron deficiency. Ferrous enzymes (nitrite and nitrate reductases) affect the NO3- absorption. In a d d i t i o n , g r o w t h o f p l a n t s d e c r e a s e s d u e t o decreased NO3- absorption and the synthesis of metabolites (proteins, nucleic acids, chlorophylls, etc.) (Borlotti et al., 2012). The use of chelating agents is useful to remove iron uptake problems by plants (Abadía et al., 2011). Magnetic fluids at suit- able concentrations have a positive effect on the photosynthetic capacity of plant. Iron ions in the structure of magnetic fluids can be an important source of iron for the development of plants (Răcuciu and Creangă, 2007 b). Iron is not in the chl structure, but it is one of the essential elements to synthesize chl (Răcuciu et al., 2009). In general, magnetic fluids are nanoparticles dispersed in water or in a hydrocar- bon fluid such as citrate. Biocompatibility of mag- netite (Fe3O4) has been confirmed (Răcuciu et al., 2009). However, treating plants with electromagnet- ism or any other magnetic field with these particles is destructive and changes the genotype and pheno- type of plants and causes chromosomal deviations (Pavel et al., 1999; Pavel and Creangă, 2005; Răcuciu and Creangă, 2007 a, b; Răcuciu et al., 2009). Chitosan is one of deacetylated derivatives of chitin. As a natural polymer is abundant and can be degraded by biological agents and it can be used in agriculture. This molecule is environmentally friendly and non-toxic that is used in the formulation of slow release fertilizers (Wu and Liu, 2008). In vitro use of chitosan in a suitable concentration in Vitis vinifera L. Hatamzadeh and Shafiei-Masouleh - Organic fertilizer for Lilium 217 stimulated the photosynthesis and increased plant growth with increasing root and shoot biomasses. It also protected the plant against Botrytis cinererea fungus and cytological changes (Barka et al., 2004). We utilized the effects of chitosan, as nanoparticle with magnetic properties on plant growth and devel- opment in lily bulb production (Shafiee-Masouleh et al., 2014) and cucumber (the data were not pub- lished yet). Modern agriculture should look for factors that while having positive effects as well as its use to be easy, affordable and reasonable. It is not possible to use a large magnetic field in farms. Also, use of macromolecule of chitosan as foliar application will cause stomata obstruction and reduce gas exchange and photosynthesis. Therefore, the achievement of a soluble compound or nano-structured composite with the synergic effects of two compounds (mag- netite and chitosan) will be very valuable at the same time. To achieve these purposes, this research stud- ies the following: i) Increasing yield and post-harvest quality of cut lily by increasing the efficiency of nutri- ent uptake and photosynthetic performance of the plant; ii) Institutionalization of chitosan use at the nanoparticle scale during forcing of lily flowers; iii) Investigating the effect of magnetic nanoparticles on yield and quality of lily flowers; and iv) Physiological and morphological understanding of the effect of chi- tosan nanoparticles on the yield and quality cut flow- ers of lily. 2. Materials and Methods Plant material and cultivation conditions In this study, bulbs of three cultivars of Lilium, including Brunello (Asiatic, 16-18 cm in circumfer- ences), Navona (Asiatic, 16-18 cm in circumferences) and Cherbourg (Oriental, 18-20 cm in circumfer- ences) were used to study effects of two organic sup- plements of fertilizer (OSFs). After melting of vernalizing substrate of bulbs, the bulbs were disinfected with 0.1% benomyl fungicide for 15 min and dried in the air for one day. Thirty uni- form bulbs in size were selected per cultivars to force. The culture substrate contained cocopeat and perlite (1:1, v/ v) that was completely homogeneous. The bulbs were planted in one-kilogram plastic bags with some pores into 10 cm depth and irrigated with tap water until emergence. After emergence every 2- 3 days depending on the weather and substrate con- ditions were fertigated. Greenhouse experiments in the research green- h o u s e o f t h e F a c u l t y o f A g r i c u l t u r a l S c i e n c e s , University of Guilan, Rasht, Iran in latitude of 37° 12’ 3.94’ N and longitude of 49 38’ 55.78’ E were per- formed. Preparation of fertilizer supplements C a r b o x y m e t h y l a t i o n o f c h i t o s a n . T h e c a r - boxymethylated chitosan (CMC) was prepared from chitosan [poly (d-glucosamine), (C 6 H 11 NO 4 ) n , deacety- lated chitin] with low molecular weight 100,000- 300,000 (Acros, Acros Organics, Geel, Belgium) to increase solubility of chitosan by carboxymethyla- tion. The procedure of preparing of CMC was pre- sented by Shafiee-Masouleh et al. (2014). An IR (IR- 470, Shimadzu, Kyoto, Japan) (Fig. 1) was used to analyze the produced H-form CMC (not salt contain- ing Na ion). Preparation of nano-particles magnetite According to Shafiee-Masouleh et al. (2014), nano-particles of Fe3O4 were synthesized. FeCl3 (Merck, Germany) and FeCl2.4H2O (Merck, Germany) were used to prepare Fe3O4. The whole procedure of preparing Fe3O4 can be observed at the mentioned paper. Preparation of magnetic nano-carboxymethylated chitosan The magnetic nano-carboxymethylated chitosan (MNCC) was prepared with encapsulation of Fe3O4 by carboxymethylated chitosan (CMC) according to Fig. 1 - IR spectrum of carboxymethylated chitosan. This spec- t r u m s h o w s t h a t t h e c a r b o x y l m e t h y l g r o u p s (-CH3COOH; 1741 cm-1; stretching vibration feature of C=O groups) resulted from monochlroacetic acid salt are linked to O atom in -OH group, and -NH2 exists on car- boxymethylated chitosan. Adv. Hort. Sci., 2019 33(2): 215-226 218 Shafiee-Masouleh et al. (2014). This was carried out by carbodimiide (Merck, Germany) and saline phos- phate buffer in sonication conditions. The details of laboratory procedures can be studied in the men- tioned paper. Analyses of the size and morphology of nano-particles were performed with SEM (Fig. 2), and IR and XRD were used to identify the coating and structure of MNCC (Figs. 3 and 4). Statistical design A split plot factorial experiment was conducted in a completely randomized design with 15 main plots and 9 (1 + 8) sub plots. The main factors included three Lilium cultivars and sub factors were two types of compounds as organic supplements of fertilizer (OSFs); including carboxymethylated chitosan (CMC) and magnetic nano-carboxymethylated chitosan (MNCC) both of them at concentrations of 0, 2.5, 5, 10 and 15 mg/l. One group of zero concentration was considered as control treatment for both of com- pounds. The experiments were carried out by 5 repli- cations and with 135 pots in the research green- h o u s e o f t h e F a c u l t y o f A g r i c u l t u r a l S c i e n c e s , University of Guilan, Iran. At the end of forcing peri- od, the growth and morphological characteristics were measured for each plant (in puffy bud stage: when first flower bud showed the color and little opening in top of bud) and were harvested at a height of 45 cm from the final flower bud and trans- ferred to the laboratory. After the stems were cut diagonally under water, they were placed into a 1000 ml bottles containing 500 ml distilled water and transferred to the growth chamber to study the post- harvest life and biochemical properties. Variance analysis of data was measured using SAS software (version 9.1, 2003). Mean comparisons were performed with Tukey’s test at probability lev- els of 5 or 1% according to variance analysis. 3. Results and Discussion Magnetic nanoparticles (Pavel et al., 1999; Pavel and Creangă, 2005; Răcuciu and Creangă, 2007 a, b; Răcuciu et al., 2009) in maize and barley, constant magnetic field (Dhawi et al., 2009) in a palm tree, pulsed magnetic field (Radhakrishnan and Kumari, 2012) in soybeen, chitosan (Górnik et al., 2008) in grape and chitosan oligosaccharides (Dzung et al., 2011) in coffee seedlings have been investigated. All of researchers reported an increase in mineral uptak- Fig. 2 - The image of magnetic nano-carboxymethylated chito- san particles by SEM microscopy. Size and morphology of Fe3O4 coated by carboxmethylated chitosan. Fig. 3 - IR spectrum of magnetic nano-carboxymethylated chito- san. Transferring the absorbance band of -NH2 group from 1589 cm-1 to 1602 cm-1 and increasing absorbance intensity in wavelength number 1741 cm-1 show ester bond into some parts of carboxy groups in carboxy- methyl chitosan on the surface of Fe3O4 nanoparticles. Fig. 4 - XRD pattern of magnetic nano-carboxymethylated chi- tosan. The widths of large peaks report crystal proper- ties of magnetic nanoparticles. The average of particles sizes based on Debye Scherrer equation was calculated 10 nm. Hatamzadeh and Shafiei-Masouleh - Organic fertilizer for Lilium 219 Unlike variance analysis, significant differences between three factors were observed on the vegeta- tive length, total length and chl intensity. Differences between treatments on vegetative development showed that morphological characteristics are less affected by the experimented OSFs and their concen- trations or these compounds may have decreasing effects on these characteristics. In Cherbourg cultivar, the highest length of vegetative stem was observed in the absence of OSFs or at low concentration (5 mg/l) of CMC, which showed a significant difference with application of 2.5 mg/l CMC in Navona. Of course, these differences were not significant com- ing by the effects of magnetism or chitosan, sepa- rately. Based on the results of 3-way ANOVA test (Table 1), effects of OSFs on vegetative development of three lily cultivars due to the uniform variances of data around the mean and Type I Error did not show a significant differences for 3-way interactions between cultivars, compounds and their concentra- tions. Only a significant 2-way interaction (between cultivar and concentrations of OSFs) was observed on the chl intensity by ANOVA test. And also, it was observed significant differences between cultivars (Table 1). N=5. The means with similar letters have not any significant differences at HSD0.01. W HSD0.05 y Type I Error **, *, NS = Significance at p≤0.01 and p≤0.05 and non-significance, respectively. Table 1 - Effects of OSFs on vegetative development of three lily cultivars Cultivars (A) Treatments Vegetative length (cm) Total length (cm) w Chl intensity (SPAD index)OSFs (B) Cons of OSFs (mg/l) (C) Cherbourg CMC 0 56.08±2.39 a 84.16±2.51 a 46.62±1.76 efg 2.5 54.48±5.14 ab 81.36±5.40 ab 48.52±2.43 defg 5 57.70±2.14 a 84.60±3.52 a 42.46±2.74 ef 10 52.30±3.31 ab 79.80±3.98 ab 42.16±2.22 ef 15 54.10±1.49 ab 81.40±3.34 ab 46.70±2.35 efg MNCC 0 56.08±2.39 a 84.16±2.51 a 46.62±1.76 efg 2.5 55.30±2.91 ab 82.00±3.29 ab 46.50±3.21 efg 5 53.00±3.41 ab 81.40±4.41 ab 46.32±1.99 efg 10 53.12±1.36 ab 80.60±1.93 ab 39.98±2.83 g 15 50.84±2.73 ab 82.20±2.65 ab 46.02±1.99 efg Navona CMC 0 48.30±2.23 ab 81.20±3.01 ab 59.52±0.31 abcd 2.5 47.60±2.01 b 78.00±1.92 ab 60.16±1.54 abcd 5 50.80±2.00 ab 81.80±1.57 ab 61.60±0.58 abc 10 50.30±1.51 ab 81.90±2.65 ab 61.98±1.15 ab 15 46.20±1.83 ab 76.00±2.07 ab 61.40±1.57 abc MNCC 0 48.30±2.23 ab 81.20±3.02 ab 59.52±0.31 abcd 2.5 39.60±0.80 ab 69.90±0.48 ab 57.10±3.82 abcde 5 49.30±2.22 ab 80.00±1.25 ab 60.16±1.13 abcd 10 45.60±1.83 ab 75.30±2.33 ab 62.72±1.83 a 15 50.10±1.86 ab 80.00±3.58 ab 57.86±0.97 abcde Brunello CMC 0 50.90±2.24 ab 70.70±2.29 ab 53.38±3.54 abcdefg 2.5 49.10±4.14 ab 68.50±5.34 ab 51.10±2.14 abcdefg 5 50.90±3.12 ab 69.20±4.53 ab 50.12±1.74 abcdefg 10 52.60±4.01 ab 72.30±5.27 ab 53.76±1.81 abcdef 15 49.50±1.90 ab 66.80±2.81 b 49.34±0.50 bcdefg MNCC 0 50.90±2.24 ab 70.70±2.89 ab 53.38±3.54 abcdef 2.5 54.50±2.23 ab 77.30±1.72 ab 49.70±0.61 bcdefg 5 49.90±2.14 ab 69.80±2.18 ab 51.84±1.78 abcdefg 10 50.90±3.60 ab 69.60±3.14 ab 51.60±1.68 abcdefg 15 52.80±3.18 ab 71.80±3.99 ab 49.22±2.08 cdefg ANOVA (3-way) A ** ** ** B NS NS NS C NS NS NS A×B NS NS NS A×C NS NS * B×C NS NS NS A×B×C NS y NS y NS y CV (%) 10.46 8.68 8.58 220 Adv. Hort. Sci., 2019 33(2): 215-226 pared to other treatments. Total length showed simi- lar reaction with vegetative length to treatments but the least value was observed in Brunello treated by 15 mg/l CMC. However, chl intensity under effects of treatments showed the most value in Navona treated by 10 mg/l MNCC and the least were observed in Cherbourg treated by 10 mg/l MNCC (Table 1). It seems that morphological characteristics of cultivars treated by two types of OSFs in different concentra- tions are under dominant effect of cultivar; on the other hand, morphological characteristics of different lily cultivars show the various reactions to OSFs and their concentrations. Generally, from the above observations on vegetative development, it can be interpreted that firstly, size of the plant is affected by genotype, and secondly, magnetism may increase the synthesis of chl and chl density, but it does not affect the size of the shoots and this needs to be investigat- ed more with higher concentrations. Nguyen Van et al. (2013) stated that chitosan nanoparticles can easily penetrate into plant cells and increase the biological activity of cells. They pointed to an important effect of chitosan nanoparti- cles on the biophysical properties of the coffee plant such as increasing chl content and plant growth and development. Răcuciu and Creangă (2007 b) used low-density (less than 100 μl/l) ferrofluids without electromagnetic treatment or any other magnetic field for corn seedlings. They observed that due to their super-paramagnetic effects (a special effect of magnetic nanoparticles) on the structure of photo- synthetic enzymes, growth was enhanced. Răcuciu and Creangă (2007 a, b) and Răcuciu et al. (2009) used two fluids of magnetic nanoparticles of Fe3O4 coated with or without tetraethylammonium hydrox- ide in Petri dishes for germination of corn seeds with- out the use of a magnetic field (50 μl per liter). They observed that plant height increased, but when the concentration increased, toxicity was reported. They referred that magnetic iron is a source for plant growth. In addition, magnetism has a good effect on photosynthesis. El-Sayed (2014) reported the increas- ing effect of magnetic water treatment on plant growth of Vicia faba, chl a and b, and carotenoids, and contents of gibberellic acid and kinetin It is known that kinetin (cytokinins) is effective in preser- vation and prevention of the chlorophylls destruction (Taiz and Zeiger, 2002). Farouk and Amany (2012) reported an increase in chl a and total chl by spraying the chitosan (250 mg/ l) on chickpea compared to low concentrations (50 a n d 1 2 5 m g / l ) , a n d t h e i r i n t e r p r e t a t i o n a b o u t increasing the content of photosynthetic pigments was the increase of cytokinin level and stimulation of the synthesis of chlorophyll, and they interpreted this as the role of amino groups in chitosan on the men- tioned processes. Chlorophyll density below 40 (Spad index) indicates a disruption to photosynthetic process (Netto et al., 2005). However, health of the photosynthetic apparatus in all treatments, especial- ly high concentrations of two OSFs (magnetic and non-magnetic) can be seen. Răcuciu et al. (2009) reported that when concentration of Fe3O4 magnetic nanofluid was increased up to 300 μl/l, content of chl a decreased by about 20 mg/l of fresh weight. In addition, Răcuciu and Creangă (2007 b) stated that the magnetic nanofluid of Fe3O4 coated with tetram- ethylammonium hydroxide (50 μl per liter) produced more pigments (chl a and b and carotenoid), and higher concentrations of fluids destroyed the photo- synthetic process. Limpanavech et al. (2008) report- ed that chitosan affected the expression of chloro- plast genes in Dendrobium orchid and altered the chloroplast size. Dzung et al. (2011) sprayed chitosan at appropriate concentrations on coffee seedlings and reported an increase in content of chlorophyll and also in uptake of mineral elements (nitrogen, phosphorus, potassium, calcium and magnesium), and enhancement of photosynthesis as well as the synthesis of chlorophyll of leaves. As can be seen in Table 2, 3-way interactions between cultivars, OSFs and their concentrations in ANOVA test due to Type 1 Error could not affect reproductive development of lily plants. However, mean comparisons showed significant effects at HSD0.01 on these characteristics. In Cherbourg, the most inflorescence lengths were observed in the plants treated with 15 mg/l MNCC, but in Navona without significant different with Cherbourg, the most lengths of inflorescence was observed in con- trol and 10 mg/l CMC. The shortest length of inflores- cence was observed in Brunello. However, this culti- var with the least concentration of magnetic OSF had the most length and generally, it seems that on this characteristic, the effect of genotype was dominant toward OSFs and their concentrations. How to make a reaction of a plant as flower bud number affected by 3-way interactions of factors shows dominant effects of genotype toward OSFs and their concentra- tions. We can statistically analyze each cultivar sepa- rately to observe 2-way interactions and/or single effects of OSFs on the responses of lily plants. Cherbourg and Navona could not show response to OSFs and their concentrations for bud number. Hatamzadeh and Shafiei-Masouleh - Organic fertilizer for Lilium 221 However, Brunello reaction to OSFs and their con- centrations was obvious. It can be said that this culti- var could respond to the OSFs compared with two other cultivars, because produced the most bud number with 2.5 mg/l of magnetic compound. In the case of bud length, although 3-way interaction showed significance, it can be seen for this morpho- logical characteristic that the effect of genotype was dominant again, and Cherbourg produced larger buds c o m p a r e d w i t h t w o o t h e r s . F u r t h e r m o r e , t h i s response was observed about flowering date, and Cherbourg flowered later (Table 2). From the obser- vations of the effects of three factors, including culti- var, OSFs and concentrations of them, it can be inter- preted that this range of the tested concentrations could not affect vegetative and reproductive mor- phology, and it seems that more concentrations of OSFs must be investigated in future research. It is known that one of the factors that contribute to growth of the reproductive part (flower bud length) is potassium. Amino groups in the car- boxymethylated chitosan and its derivatives can Table 2 - Effects of OSFs on reproductive development of three lily cultivars Cultivars (A) Treatments Inflorescence length (cm) Bud No. Bud length (mm) Flowering date (days)OSFs (B) Concentration of OSFs (mg/l) (C) Cherbourg CMC 0 28.08±0.56 abc 6.80±0.58 abc 116.63±0.24 a 94.60±0.24 a 2.5 26.88±0.84 abcdef 6.80±0.37 abc 116.31±0.77 a 93.00±0.77 a 5 26.90±1.57 abcdef 6.20±0.20 b 123.27±0.49 a 93.20±0.49 a 10 27.50±1.47 abcd 7.00±0.32 abc 116.14±0.55 a 92.5±0.55 a 15 27.30±1.87 abcde 6.40±0.40 abc 118.64±0.37 a 93.80±0.37 a MNCC 0 28.08±0.56 abc 6.80±0.58 abc 116.64±0.24 a 94.60±0.24 a 2.5 26.70±1.09 abcdef 6.40±0.51 abc 122.06±0.51 a 93.60±0.51 a 5 28.40±1.41 ab 6.80±0.37 abc 120.39±0.58 a 93.80±0.58 a 10 27.48±0.88 abcd 6.80±0.37 abc 110.43±0.68 a 93.40±0.68 a 15 31.36±2.06 a 7.00±0.58 abc 119.86±0.81 a 94.40±0.81 a Navona CMC 0 32.90±1.61 a 9.00±0.32 a 82.87±0.68 b 59.40±0.68 b 2.5 30.40±0.53 ab 9.50±0.29 ab 80.02±0.73 b 60.20±0.73 b 5 31.00±2.26 ab 9.20±0.49 a 79.80±0.49 b 59.80±0.49 b 10 31.60±1.35 a 9.00±0.63 a 81.16±0.63 b 59.00±0.63 b 15 29.80±1.11 ab 9.40±0.24 a 78.72±0.49 b 58.80±0.49 b MNCC 0 32.90±1.61 a 9.00±0.32 a 82.87±0.68 b 59.40±0.68 b 2.5 30.30±0.89 ab 9.20±0.97 a 76.04±0.58 b 59.20±0.58 b 5 30.70±1.07 ab 9.00±0.32 a 77.69±0.60 b 59.60±0.60 b 10 29.70±1.07 ab 10.00±0.71 a 78.95±1.02 b 59.20±1.02 b 15 29.90±2.34 ab 8.40±0.68 a 80.34±0.60 b 59.60±0.60 b Brunello CMC 0 19.80±1.41 cdefg 5.40±0.60 c 78.91±0.97 b 60.20±0.97 b 2.5 19.40±1.37 defg 5.80±0.97 b 86.39±1.47 b 61.60±1.47 b 5 18.30±1.52 g 5.80±0.66 b 79.46±1.71 b 62.20±1.71 b 10 19.70±1.38 defg 5.80±0.97 b 80.04±1.56 b 62.80±1.56 b 15 17.30±1.28 g 5.60±1.20 c 84.75±1.38 b 61.00±1.38 b MNCC 0 19.80±1.41 cdefg 5.40±0.60 c 78.91±0.97 b 60.20±0.97 b 2.5 22.80±1.23 bcdefg 6.40±0.75 abc 84.40±1.59 b 62.20±1.59 b 5 19.90±0.89 cdef 5.40±0.75 c 78.47±1.17 b 60.60±1.17 b 10 18.70±0.85 fg 4.60±0.24 c 76.75±0.20 b 61.20±0.20 b 15 19.00±0.96 g 5.50±0.96 b 80.59±1.72 b 61.60±1.72 b ANOVA (3-way) A ** ** ** B NS NS NS C NS NS NS A×B NS NS NS A×C NS * ** B×C NS NS NS A×B×C NS y NS y NS y CV (%) 17.61 6.69 1.63 N=5. The means with similar letters have not any significant differences at HSD0.01. W HSD0.05 y Type I Error **, *, NS = Significance at p≤0.01 and p≤0.05 and non-significance, respectively. Adv. Hort. Sci., 2019 33(2): 215-226 222 serve as a place for the absorption of some metal cations. This compound is used to remove heavy metals and water purification and this shows its chelating role (Chang and Chen, 2005; Chang et al., 2006). Chelating sites of chitosan may be effective in absorbing essential metals ions such as manganese, iron (Fe+2 and Fe+3), potassium, magnesium, etc. for plants. The abilities of polymer or oligomer chitosan to stimulate plant growth under in vitro conditions; V i t i s v i n i f e r a L . ( B a r k a e t a l . , 2 0 0 4 ) ; o r c h i d Dendrobium phalaenopsis (Nge et al., 2006); and the growth of protocorm like bodies in the Dendrobium orchid (Pornpienpakdee et al., 2010) have already been reported. Pornpienpakdee et al. (2010) used chitosan macromolecules, which had been deacety- lated up to 70%, at a concentration of 10 mg/l under in vitro culture conditions for Dendrobium orchid and produced plants with more length. In addition, Limpanavech et al. (2008) when used the polymer or oligomer chitosan (with deacetylated degrees of 70, 80 or 90%) at concentrations of 1-100 mg/l, they observed that polymers at concentrations of 1-10 mg/l and oligomers at concentrations of 50-100 mg/l resulted in more inflorescences production. Gornik et al. (2008) applied a type of commercial compound of chitosan, Biochikol 02.PC containing 2% chitosan, at concentration of 0.5% on grape cuttings and report- ed that root system expanded and it increased the n u m b e r o f n e w b r a n c h e s a n d t h e i r l e n g t h s . Radhakrishnan and Kumari (2012) reported an increase in leaf number and growth of soybean, and an increase in pod length and grain weight under the influence of pulsed magnetic field. El Sayed (2014) reported that magnetic water treatment increased the sink yield in bean (seed and number of seeds per plant). He suggested this effect may be due to increased photosynthetic function of the plant under influence of magnetic treatment. Ohta et al. (1999) reported an increase in the photosynthetic reservoir (flower number) in lisianthus with plant seed treat- ment by chitosan acid solution. Limpanavech et al. (2008) also demonstrated that mechanism of chi- tosan effect on increasing the number of plant pho- tosynthesis reservoirs (number of flowers) in the Dendrobium orchid may be caused by effect on the development of photosynthetic apparatus and increasing the size of chloroplasts after chitosan spraying. Kananont et al. ( 2010) reported that differ- ent types of chitosan as poly- and oligosaccharides with varying degrees of deacetylation at a concentra- tion of 10 mg/l are effective for in vitro culture medi- um of seed germination of Dendrobium orchids and growth of the protocorm like bodies. They stated that chitosan amino groups may be effective factor o n t h e g r o w t h o f t h e p r o t o c o r m l i k e b o d i e s . Pornpinpakdee et al. (2010) described role of con- centration of different types of chitosan on growth rate of orchid Dendrobium. The results of 3-way ANOVA (Table 3) show signif- icant differences in content of soluble carbohydrates of terminal bud of inflorescence in three cultivars of lily treated with OSFs and their concentrations. The highest content of glucose was significantly observed in Brunello treated with 15 mg/l OSFs regardless of their magnetic or non-magnetic properties. Navona was in second rank in response to treatments com- pared to Brunello. However, Cherbourg under effects of OSFs and their levels showed the least glucose content regardless of being magnetism or not com- pared with two other cultivars. Fructose synthesis in the terminal bud had different reaction to the type of OSFs based on genotypes. Generally, the most con- centrations of OSFs regardless of their structures had the most effect on fructose content (Table 3). Sucrose approximately had similar response to glu- cose toward treatments, but about this carbohydrate after ‘Brunello’, ‘Cherbourg’ had second score for the most content not ‘Navona’. Starch and other sugars and proteins (enzymes) are photosynthetic products that are transported to storage organs (flowers, seeds or bulbs). Iron acts as a c o f a c t o r f o r m a n y e n z y m e s , f o r m i n g p a r t o f cytochromes and involves in biochemical reactions, including respiration, photosynthetic material trans- fer, nitrate synthesis, nitrogen fixation, and DNA syn- thesis (He et al., 2011). El Sayed (2014) reported that photosynthetic function of bean plant increases with magnetic water treatment. He reported that con- tents of glucose and sucrose as well as polysaccha- rides in the leaves, stems and the whole plant of bean were higher toward irrigation without magnetic field. In addition, positive effect of chitosan on plant growth may be due to its effect on increasing the phosphorus content. Phosphorus is an essential ele- ment in the biosynthesis and carbohydrate transfer for cell division and the formation of DNA and RNA (Farouk and Amany, 2012). According to Table 4, variance analysis of OSFs effects (type and concentration) on the α-amylases activities of three cultivars of lily showed the signifi- cance of 3-way interactions. OSFs effects on α-amy- lase in ‘Navona’ compared with two other cultivars showed the highest activity of this enzyme in the highest tested concentration of non-magnetic OSF. Hatamzadeh and Shafiei-Masouleh - Organic fertilizer for Lilium 223 While, the highest activity recorded for this enzyme in ‘Brunello’ was observed in magnetic OSF (MNCC) unlike ‘Navona’. Cultivar Cherbourg unlike two other cultivars showed the highest activity of α-amylase regardless of being magnetic of OSF with the high concentrations of two OSFs, in tissue of the last flower bud of inflorescence. Therefore, responses types of cultivars toward being magnetic or not of OSFs were different. On the other hand, the amount of activity of this enzyme in ‘Navona’ was higher regardless of the type of OSF and their concentra- tions. According to 3-way ANOVA, β-amylase activity in tissue of the terminal flower bud of inflorescence was significantly affected by three factors (Table 4). The activity of this enzyme unlike α-amylase was more in ‘Brunello’, and like α-amylase, the activity of this enzyme was more with the highest concentra- tion of magnetic OSF. The β-amylase activity in ‘Cherbourg’ was like α-amylase in the same cultivar. However, in ‘Navona’, this enzyme showed the high- est activity affected by the both of magnetic and Table 3 - Effects of OSFs on soluble carbohydrates into the terminal bud tissue of inflorescence in three lily cultivars N=3. The means with similar letters have not any significant differences at HSD0.01. W HSD0.05 y Type I Error **, *, NS = Significance at p≤0.01 and p≤0.05 and non-significance, respectively. Cultivar (A) Treatments Glucose Fructose Sucrose OSFs (B) Concentration of OSFs (mg/l) (C) Cherbourg CMC 0 0.80±0.11 g 2.20±0.13 ghij 3.21±0.19 ghi 2.5 1.95±0.24 fg 2.21±0.18 ghij 5.70±0.28 def 5 3.54±0.18 de 3.34±0.17 efg 8.21±0.35 ab 10 4.50±0.23 bcd 3.33±0.20 efgh 8.19±0.28 abc 15 3.50±0.24 de 4.61±0.21 bcd 5.70±0.23 def MNCC 0 0.80±0.11 g 2.20±0.13 ghij 3.21±0.19 gh 2.5 0.89±0.13 g 3.33±0.16 efghi 5.71±0.24 def 5 1.98±0.23 fg 3.27±0.18 efghi 5.67±0.15 def 10 3.50±0.11 de 4.60±0.28 bcd 8.23±0.28 ab 15 2.01±0.29 g 5.51±0.23 ab 8.22±0.44 ab Navona CMC 0 2.64±0.22 ef 2.81±0.18 fghi 1.53±0.26 i 2.5 2.66±0.24 ef 4.03±0.24 cde 3.03±0.20 hi 5 3.49±0.22 de 4.03±0.20 cde 3.07±0.19 hi 10 5.03±0.31 abc 4.83±0.26 bc 4.53±0.25 fgh 15 5.02±0.28 abc 6.05±0.31 a 5.81±0.17 de MNCC 0 2.64±0.22 ef 2.81±0.18 fghi 1.53±0.26 i 2.5 3.51±0.23 de 1.54±0.13 jk 1.59±0.15 i 5 5.02±0.32 abc 2.60±0.11 fghij 3.10±0.16 ghi 10 5.84±0.23 ab 2.75±0.19 fghi 3.06±0.19 hi 15 5.88±0.23 ab 4.02±0.17 cde 4.49±0.23 fgh Brunello CMC 0 1.54±0.17 fg 0.82±0.12 k 4.73±0.23 fgh 2.5 2.72±0.18 ef 0.83±0.14 k 6.80±0.34 bcd 5 4.03±0.25 cde 2.12±0.14 ij 06.80±0.31 bcd 10 4.81±0.23 abcd 3.65±0.19 cdef 9.81±0.41 a 15 6.03±0.30 a 3.57±0.19 def 9.84±0.46 a MNCC 0 1.54±0.17 fg 0.82±0.12 k 4.73±0.23 efg 2.5 4.02±0.23 cde 0.88±0.18 k 4.81±0.24 efg 5 4.81±0.25 abcd 2.13±0.18 hij 6.46±0.19 cde 10 4.84±0.21 abcd 3.58±0.20 def 9.82±0.40 a 15 6.02±0.26 a 4.70±0.25 bcd 9.79±0.36 a ANOVA (3-way) A ** ** ** B NS ** ** C ** ** ** A×B ** ** ** A×C ** ** ** B×C NS NS ** A×B×C ** ** ** CV (%) 10.88 10.28 7.96 Adv. Hort. Sci., 2019 33(2): 215-226 224 non-magnetic OSFs with different manner toward α- amylase in the same cultivar. The Earth’s magnetic field affects orientation of the ferromagnetic particles and modulations of reac- tions. It has been reported that the magnetic field has an effect on the biochemical processes and stimula- tion of the activity of proteins and enzymes on increase of the seed vigor (Dhawi et al., 2009). Tham et al. (2001) reported an increase in shoot growth of rice seedling in the hydroponic medium using acid solution of chitosan. Magnetic fields have been reported to increase sugar content in sugar beet roots (Beta vulgaris) and the content of gluten in wheat (Triticum aestivum) (Dhawi et al., 2009). Radhakrishnan and Kumari (2012) in their experiment on soybeans indicated a positive effect of a pulsed magnetic field on the increased activity of the α- and β-amylases. In our experiment, MNCC with its syner- getic effects of magnetism, chitosan, and iron, was remarkably increased the photosynthetic structures, Table 4 - Effects of OSFs on amylases (N=3) into the terminal bud tissue of inflorescence and vase life (N=5) of three lily cultivars z The means with similar letters have not any significant differences at HSD0.01. W HSD0.05 and N=5 y Type I Error **, *, NS = Significance at p≤0.01 and p≤0.05 and non-significance, respectively. Cultivars (A) Treatments α-amylase β-amylase Vase life (days)wz OSFs (B) Concentration of OSFs (mg/l) (C) Cherbourg CMC 0 4.48±0.21 gh 5.98±0.49 g 7.00±0.00 ab 2.5 4.42±0.21 gh 7.97±0.30 g 8.20±0.97 ab 5 6.61±0.33 ef 12.31±0.90 de 7.00±0.71 ab 10 6.61±0.20 ef 13.49±0.68 d 7.20±0.37 ab 15 9.00±0.32 cd 12.33±0.99 de 6.00±0.63 ab MNCC 0 4.48±0.21 gh 5.98±0.49 g 7.00±0.00 ab 2.5 6.56±0.21 ef 6.01±0.43 g 6.60±0.51 ab 5 7.14±0.17 def 8.04±0.30 g 6.67±0.67 ab 10 8.99±0.49 cd 12.04±0.95 def 8.00±0.00 ab 15 11.07±0.44 ab 8.07±0.33 g 6.40±0.51 ab Navona CMC 0 5.60±0.27 fg 9.06±0.36 efg 7.80±0.37 ab 2.5 8.09±0.40 cde 9.11±0.45 ef 7.20±0.49 ab 5 8.02±0.41 cde 12.15±0.63 de 7.80±0.20 ab 10 9.63±0.34 bc 15.21±0.65 cd 8.60±0.51 a 15 12.04±0.53 a 15.11±0.73 cd 8.20±0.49 ab MNCC 0 5.60±0.27 fg 9.06±0.36 efg 7.80±0.37 ab 2.5 3.07±0.20 hi 12.10±0.50 def 6.80±0.37 ab 5 5.57±0.22 fg 14.92±0.68 cd 8.80±0.58 a 10 5.59±0.27 fg 18.09±0.79 abc 7.80±0.37 ab 15 8.03±0.34 cde 18.13±0.74 abc 9.25±0.48 a Brunello CMC 0 2.04±0.14 i 6.52±0.37 g 5.80±0.58 ab 2.5 2.18±0.12 i 8.16±0.44 fg 4.80±0.73 b 5 4.27±0.24 gh 13.38±0.53 d 6.20±0.37 ab 10 6.82±0.32 ef 15.27±0.75 cd 7.00±0.77 ab 15 6.81±0.35 ef 19.30±0.69 ab 7.67±0.67 ab MNCC 0 2.04±0.14 i 6.52±0.37 g 5.80±2.58 ab 2.5 2.09±0.07 i 12.99±0.61 de 6.20±0.20 ab 5 4.18±0.22 gh 15.43±0.81 bcd 7.60±0.51 ab 10 6.80±0.24 ef 15.11±0.70 cd 5.80±1.02 ab 15 9.29±0.50 bc 19.41±0.79 a 5.80±1.02 ab ANOVA (3-way) A ** ** * B ** * NS C ** ** NS A×B ** ** NS A×C ** ** NS B×C ** ** NS A×B×C ** ** NSy CV (%) 7.84 7.99 19.18 Hatamzadeh and Shafiei-Masouleh - Organic fertilizer for Lilium 225 the transfer of photosynthetic products and the stor- age of carbohydrates; so that Takeda et al. (1983) stated that the increase of substrate promotes the activity of amylase enzymes, especially α-amylase. According to Table 4, the longer vase life was observed in ‘Navona’ in the high concentrations of OSFs (magnetic and non-magnetic) based on 3-way interactions compared with two other cultivars. Hajnorouzi et al. (2011) reported that using a combi- nation of Earth’s magnetic field and weak pulsed electromagnetic field on corn seedlings can increase the growth rate and decrease the iron content of the plant and maintain the membrane’s health and decrease oxidative burst. Since the magnetic field is the natural property of the earth, plants and other living creatures are per- manently responding to the magnetic field during their lives. Earth acts as a magnet with its northern and southern poles, and natural effects of the mag- netic field can change the growth and yield of plants on the ground. In particular, the electromagnetic spectrum of solar radiation stimulates plant growth through the process of photosynthesis. The possible mechanism is the changing of the electrostatic bal- ance of the plant system at the membrane surface of the cell, which is the primary site for any plant growth restriction or promotion (Radhakrishnan and Kumari, 2012). Therefore, the role of superparamag- netic nanoparticles can be interpreted in our experi- ment based on the influence of the Earth’s magnetic field. It can be stated, instead of the iron salt (which is often difficult to uptake by plants), a chelating agent in the nutrition solution can be used both as the iron source for the plant and also is suitable for the absorption of other elements. Between two tested compounds, MNCC is a more suitable compound than CMC because it has a positive synergic effect in addition to providing iron in the plant, which is effect of both chitosan and magnetism. In addition, the characteristics of particle size in this type of supple- ment, i.e. nano- size, affect the biophysical character- istics and biological activities of the plant. 4. Conclusions Our experiment showed that utilizing the magnet- ic composite of chitosan (MNCC) or modified and chelating macromolecule of chitosan (CMC) in horti- culture can significantly affect the growth and devel- opment physiology of plants. We introduced this compound, especially magnetic compound, as fertil- izer supplement in production period of lily bulb (Shafiee-Masouleh et al., 2014); now in this experi- ment for three cultivars forcing period of lily shows that high concentration of both OSFs can be used as a supplement in nutrition solution. The highest con- centrations (10 and 15 mg/ l) regardless of OSF types and the cultivars response caused significant physio- logical effects on the content of carbohydrates and also the enzymes that are influence on carbohydrates (amylases). 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