Responses of different quality parame ters of Chia to arbuscular mycorrhiza and plant growth regulator

Field experiment was conducted to evaluate the influence of arbuscu­ lar mycorrhiza fungi (AMF) and foliar spray of plant growth regulators (PGRs) and their interaction on vegetative growth, seed yield and yield attributes and some biochemical criteria of chia (Salvia hispanica L.), in a split plot design with three replications. Plants grown in absence or presence of AMF were sprayed every 2 weeks with benzyl adenine (BA), CPPU [N­(2­chloro­4­pyridinyl)­N’­ phenylurea], common name forchlorfenuron, and Naphthalene acetic acid (NAA) at 50, 20 and 50 ppm respectively, while control plants were sprayed with tap water. The results revealed that, inoculation with AMF generally caused significant augmentation in all studied growth, yield and yield attribut­ es, total chlorophylls and carbohydrates content in leaves, augmentation in nutritional values of seeds like carbohydrates %, macronutrient, micronutri­ ents, proteins %, total flavonoids, oil % compared to non­inoculated plants. In absence or presence of AMF, application of PGRs generally caused significant increases in the studied parameters compared to control. The interaction between NAA and AMF was more effective since gave higher increases in the studied parameters. It can be concluded that, cultivation of chia plant in pres­ ence of mycorrhiza with foliar application of NAA at 50 ppm is recommended for enhancing growth, and nutritional values of seed yield.


Introduction
Salvia hispanica, ordinarily known as chia, is an annual herbaceous plant which belongs to Lamiaceae family. It is native to southern Mexico and Northern Guatemala; the word of Chia comes from the Nahuatl word "chian" with means oily. The name Salvia hispanica was specified by the Swedish botanist Carl Linnaeus, who discomfited the wildgrowing plant coming from the new world with a regional plant from Spain. It grows up to 1m tall with leaves of about 48 cm long and 36 cm wide. Chia flowers are white or purple containing oval seed mottlecolored with brown, gray, black, and white with size ranging from 1 to 2 mm. it grows naturally in tropical and subtropical environments; it is optimally established from 400 to 2500 m and considered to be a shortday plant with a threshold of 1214 h. Chia acquired acceptance owing to the high nutritional value of its seeds. The main components of seeds are polyunsaturated fatty acids Omega3 (PUFA ω3) (5864% of total lipids) and Omega6 (ω6), protein with a ratio (1624%), carbohydrates (26 41%) lipids (3135 %), and fiber (3456 %). in addition to some minerals, vitamins and high level of antioxi dants (Baginsky et al., 2016;Sosa et al., 2016;Marcinek and Krejpcio, 2017).
Arbuscular mycorrhiza fungi (AMF) are a category of soil microorganisms which form a symbiotic asso ciation with plants. Not only it could enhance uptake of mineral elements and water by plants that pro mote plant growth, but also increase crop yield, qual ity properties and active ingredients (Dupre et al., 2008;Singh et al., 2010). The positive effect of inocu lation with AMF on physiological and biochemical changes in different medicinal and aromatic plants has been reported in a number of species such as enhancing growth performance and seed yield (Gashgaril et al., 2020;Bilalis et al., 2020), increase photosynthetic pigments and carbohydrates content (Amiri et al., 2017), increased nutrient status in plant orangs (Chaudhary et al., 2008), protein content (Ouzounidou et al., 2015), antioxidants activity (Golubkina et al., 2020), fixed oil (Moghith, 2019), promoted concentration of essential oil (Chaudhary et al., 2008;AlAmri et al., 2016), enhanced primary and secondary metabolism and bioactive compounds (Gashgaril et al., 2020 ).
Plant growth regulators (PGRs) have been defined as one of the major factors affecting plants growth and their primary and secondary metabolites, NAA is an organic compound that is synthetic plant hor mone in the auxin family. it is known to enhance cell elongation, cell division, elongation of shoot, vascu lar tissue, photosynthesis, RNA synthesis, membrane permeability and water uptake is also involved in many physiological processes such as fruit set, delayed senescence, leaf chlorophyll content, stimu lates flowering and increases yield (Davies, 1987). Foliar application of NAA on different medicinal and aromatic plants have been reported to improve growth and yield attributes, photosynthetic pig ments, total carbohydrate and oil yield (Rohamare et al., 2013), enhance nutrient status in plant orangs, essential oil %, polyphenols and flavonoids content and antioxidant activity (Atteya and El Gendy, 2018). Moreover, it has been reported for ameliorating the harmful effects of salinity (Abou Elghit, 2015). Cytokinins include benzyl adenine (BA) that promotes cellular elongation and division (Krug et al., 2006). Foliar spray of BA has been reported to increase the growth and development of medicinal and aromatic plants (Matter, 2016;Moussa, 2019). It was reported to improve photosynthetic pigments content, total carbohydrate oil percentage and oil yield (Abdel Rahman and AbdelKader, 2020), contents of macronutrient and micronutrient, total phenols and total flavones (AbdelHamid, 2020), enhance protein concentration (Prins et al., 2013) and antioxidant activity (Sidkey, 2020).
Likewise, CPPU is one of PGRs. It is a cytokinin like substance that has strong cytokinin activity by stimu lating fruit set and fruit quality, it plays a role in cell division and cell wall elongation (Nickell, 1985;Zhang and Whiting, 2011). It has been reported to improve growth and yield attributes or protein percentage of medicinal plants (Abbas and Zahwan, 2016).
Although the valuable roles of AMF and PGRs on medicinal and aromatic plants and their useful influ ence on enhancing growth and production has been formerly evaluated. However, there are not enough available data about their activity on the growth and production of Chia plants. Therefore, the objective of this study was to evaluate the impact of AMF and foliar spray of PGRs (BA, CPPU and NAA) and their interactions on vegetative growth, seed yield and yield attributes, and some chemical compositions of Salvia hispanica plants.

Materials and Methods
Open field experiment was carried out during the two successive seasons of 2018 and 2019 at Experimental area of the Ornamental Horticulture Department, Faculty of Agriculture, Cairo University, Giza Governorate (latitude 30°01'13.44"N, longitude 31°12'30.24"E, altitude 22 m a.s.l.). The aim of this work was to evaluate the effects of foliar application of different plant growth regulators (PGRs) such as 6 BA (6Benzylaminopurine) benzyl adenine, CPPU [N (2chloro4pyridyl)] and NAA (Napthaleneacetic Acid) in absence or presence of arbuscular mycor rhizal fungi (AMF) on vegetative growth, seed yield and its attributes and some biochemical parameters of Chia (Salvia hispanica L.) plant.

Experimental procedure
Seeds of Salvia hispanica plants were obtained from experimental farm of Faculty of Pharmacy, Cairo University. On 1 st October and 15 th October (in the two seasons, respectively), seeds were sown in the nursery at the Ornamental Horticulture Department, Faculty of Agriculture, Cairo University, Giza. After 45 days old from seeds sowing (on the 15 th of November, and1 st December, in both seasons, respectively), uniform seedlings, with an average plant height of 1518 cm, were transplanted in the experimental open field with a distance of 70 cm among rows, 50 cm spacing between plants in plots (3.5×3.5 m).
The physical and chemical properties of the exper imental soil were determined according to Jackson (1973), and the data are recorded in Table 1. AMF inoculum contained roots, hyphae, spores and growth media from a pot culture of onion plants colonization with Glomus mosseae NRC31 and Glomus fasciculatum NRC15, and were obtained from Agricultural Microbiology Department, National Research Center. Inoculum material consisted of 275 spores g 1 oven dry bases in addition to the coloniza tion roots pieces (the infectivity 10 4 propagola). AMF inoculation treatments was achieved by mixing 5 g of it with 10 g of chia seed before sowing and repeated monthly after transplanting by injection inoculum material into the soil in roots area from five sides at 5 g/seedling.
PGRs treatments were initiated after 15 days from the transplanting, by foliar spraying the plants every 2 weeks with Sytonine 4%® [commercial name, con sists of 6 BA (6Benzylaminopurine) benzyl adenine 4 %], Cytovac® (CPPU) and Fast tonic® [commercial name, consists of Napthaleneacetic Acid 25% + Sodium nitrophenolate 0.6%]. The three commercial products were obtained from Bio Green Company, the regional representative of the Jordanian compa ny, Elite Company for the manufacture of agricultural fertilizers development, Egypt. The concentrations of BA and NAA were 50 ppm for each one, while CPPU was applied at 20 ppm and the untreated control plants were sprayed with tap water. Freshly prepared solutions of PGRs (50 mL containing Tween 20 at 1 mL/L (0.1%) as surfactant agent) were sprayed using plastic automizer until run off point. The common horticulture practices (such as irrigation, manual weeds control, fertilization) were carried out when needed.
The experimental design was split plot design with eight treatments [2 AMF (absence or presence) x 4 PGR S concentrations (including the control)] with 3 replicates, each consisting of 16 plants (2 plants from each treatment). AMF assigned to the main plots in a randomized complete blocks design and PGRs con centrations were allocated at random in subplots.

Measurement of vegetative growth and yield parameters
Vegetative growth parameters were recorded after 90 days from transplanting (On 15 th of February to 1 st March). In both seasons plant samples were taken for measurements growth characters in terms of plant height (cm), number of leaves/plant, stem diameter (cm, at 5 cm above the soil surface), num ber of branches/plant, fresh and dry weights of the herb (g/plant), root length (cm), number of root/plant and fresh and dry weights of the roots (g/plant). Whereas, at harvesting stage yield and yield attributes including number of inflorescence/ plant, number of seeds/plant, weight of seeds (g /plant), weight of 1000 seeds (g), and seeds yield (Kg/Fed.) were recorded.
Seeds yield per feddan was obtained according to the equation:  and antioxidant activity.

Chemical analysis
Total chlorophylls in fresh leaf samples were determined by using chlorophyll meter (Model SPAD 502 Minolta Co. Japan) as described by Netto et al. (2005). The total carbohydrates content in leaves and seeds (% of dry matter) was determined in dried samples according to Dubois et al., (1956). A known weight (0.1 g) of the dried samples was completely hydrolyzed with 10 ml sulphuric acid (67%) in a test tube on a boiling water bath for one hour. The solu tion was decolorized and the filtrate was completed to 100 ml with distilled water. A known volume (1 ml) of the extract was taken in a test tube, to which 1 ml phenol solution (5%) was added, followed by 5 ml of concentrated sulphuric acid. The optical density of the resulting color was measured at 490 µm, using a spectrophotometer, against a blank reagent. The standard curve of glucose was used to calculate the total carbohydrates concentration in the extract. Dried seeds samples were digested to extract nutri ents and the extract was analyzed to determine con centrations of N, P, K, Ca, Mg (% of dry seeds), Fe and Zn (ppm) which were determined according to Estefan et al. (2013). Nitrogen concentration was determined by using the microKjeldahl method. Phosphorus was determined calorimetrically by using the chlorostannous molybdophosphoric blue colour method in sulphuric acid. Potassium was determined by using the flame photometer apparatus (CORNING M 410, Germany). The concentrations of Ca, Mg, Fe and Zn were determined using atomic absorption spectrophotometer with airacetylene and fuel (Pye Unicam, model SP1900, US). Protein content in seeds was estimated by multiplying N values by 5.71 (conversion factors). Total phenolics content was determined by using the Folin Ciocalteau's reagent colorimetric method while total flavonoids content was estimated by the aluminum chloride colorimetric method and results are expressed as milligram Catechin equivalent per gram of seeds dry weight extract (mg CE/g DW) (John et al., 2014). The antioxi dant activity of seeds extract and standard antioxi dant was assessed on the basis of the radical scav enging effect on DPPH (2, 2diphenyl1picrylhy drazyl) free radicals (BrandWilliams et al., 1995).
Fixed oil %: the clean air dried seeds of chia were separately crushed in a Willey mill, then extracted in Soxhlet apparatus, samples of 10 g of seeds were moved into Soxhlet apparatus in 100 ml of Nhexane and the extraction period extended to 6 hours (3036 syphon cycle approx.). The Nhexane extract was dried over anhydrous sodium sulfate, then filtered and the oil was obtained by distillation under vacu um. Fixed oil % was calculated according to the equa tion: Fixed oil % = Extracted fixed oil weight (g) ̸ seeds sample weight (g) ̸ × 100.

Statistical analysis
The means of all obtained data were subjected to statistical analysis of variance (ANOVA) in split plot design. Combined analysis of the two growing sea sons was carried out. Means of data were compared by using Duncan's multiple range tests at 5% level Snedecor and Cochran (1989).

Vegetative growth parameters
It is evident from data in Table 2 and 3 that under the same treatments of PGRs, chia plants grown in presence of AMF had significant increase in studied vegetative growth parameters (viz., plant height, number of leaves, stem diameter, number of branch es/plant, fresh and dry weights of herb, root length, number of roots, root fresh and dry weights) com pared to those grown in absence of AMF. The obtained increases in vegetative growth attributes due to AMF inoculation are in agreement with reports of several researches on medicinal and aro matic plants including Artemisia annua (Chaudhary et al., 2008) The stimulatory influence of AMF on vegetative growth traits could be explained by AMF form symbi otic relationship with the host increase of root sur face, which led to promote root uptake of nutrients. Thus, it could significantly augment growth parame ters of tested plant (Jianheng et al., 2016). Furthermore it was indicated that the ability of AMF to enhance the availability of essential elements macro and micronutrients in the rhizospheric soil that induce its uptake and the accumulation in plant (Gashgaril et al., 2020). Table 2 and 3 also indicate that, in absence or presence of AMF, treating plants with dif ferent concentrations of PGRs resulted in significant increase in tested vegetative growth parameters compared to control. Among PGRs, application of NAA (at 50 ppm) appeared to be the most effective one particularly in presence of AMF since recorded the highest values in most cases. The results of the pronounced increase in growth parameters due to application of NAA are in accordance with findings of previous studies on medicinal plants (Alam et al., 2012;Rohamare et al., 2013;DaneshTalab et al., 2014;Abou Elghit, 2015;Rostami and Movahedi, 2016;Dheeraj and Saravanan, 2018). Moreover, numerous studies reported increase in growth attrib utes of medicinal plants owing to either BA (Matter, 2016;Moussa, 2019;AbdelRahman andAbdel Kader, 2020 andAbdelHamid, 2020) or CPPU treat ments (Abbas and Zahwan, 2016).

Results in
The stimulation effect of NAA on morphological attributes may be due its ability to increase mem brane permeability and water uptake which accom panied by elements absorption, synthesis of chloro phyll and carbohydrates contents that achieving the highest dry weight of plants (Atteya and El Gendy, 2018).

Yield and yield attributes
Data in Table 4 revealed that under the same treatments of PGRs, the values of yield and yield attributes (viz., number of inflorescence /plant, num ber of seeds/plant, weight of seeds, weight of 1000 seeds, and seeds yield) were significantly higher in plants grown in the presence of AMF than corre sponding values in the absence of AMF. The present increase in number of inflorescence/plant due to AMF inoculation is supported by the results of previ ous report (Engel et al., 2016). Moreover, recent study (Mohamed, 2020) on Foeniculum vulgare revealed that AMF inoculation significantly increased seeds yield parameters.
Exogenous application of PGRs in absence or pres ence of AMF resulted in significant increase in yield and yield parameters compared to control. Among PGRs, NAA was the most effective one particularly when interacted with presence of AMF. In this con cern, application of NAA at 50 ppm has been report ed to increase yield attributes (Rohamare et al., 2013;DaneshTalab et al., 2014;Khudus et al., 2017;Venkatesan and Shakila, 2017). Other study (Kassem et al., 2011) found that, foliar spray of NAA at 75 mg/l or CPPU at 10 mg/l caused significant increase in yield and yield attributes of Ziziphus jujuba com pared with the control. Moreover, increases in num ber of inflorescence /plant owing to application of NAA are in accordance with findings of earlier studies (Atteya and El Gendy, 2018; Dheeraj and Saravanan, 2018). While increasing yield and its attributes due to either BA application are in harmony with the find ings of various studies (Mousa et al., 2001;Matter, 2016;AbdelRahman and AbdelKader, 2020) or CPPU (Abbas and Zahwan, 2016).
The augmentation in yield and its attributes due to PGRs treatments may be due to its role in enhanced absorption of nutrients which promoted photosynthesis rate and translocation of photosyn thates and other metabolites to the sinks that lead ing to increase yield and its attributes in present study (Alam et al., 2012).

Total chlorophylls and total carbohydrates
Data in figure 1 indicate that under the same treatments of PGRs, plants grown in the presence of AMF had significantly higher values of total chloro phylls in leaves and total carbohydrates in leaves and seeds compared to corresponding values in the absence of AMF in most cases. These results run par allel with those obtained by earlier reports on medic inal plants that reported increase in chlorophyll and carbohydrates content in plants inoculated with AMF compared to controls (Amiri et al., 2017;Gashgaril et al., 2020;Moghith, 2019;Mohamed, 2020).
Results also show that, the recorded values were significantly increased in plants grown in absence or presence of AMF as a result of spraying tested PGRs compared to control. In most cases, plants sprayed with NAA in the presence of AMF had the highest val ues of the tested components. The results of increase  Rohamare et al., 2013;Rostami and Movahedi, 2016;Venkatesan and Shakila, 2017;Atteya and El Gendy, 2018), whereas the obtained increase due to either BA treatments are in close conformity with the findings of previous reports (Matter, 2016;Moussa, 2019;AbdelHamid, 2020;AbdelRahman and AbdelKader, 2020) or CPPU treatments (Kassem et al., 2011;Abbas and Zahwan, 2016).

Content of macronutrients in seeds
Results of chemical analysis of dried seeds of Salvia hispanica plants (Table 5 and 6) disclosed that, under the same treatments of PGRs the uptake and accumulation of macronutrients in seeds of plants inoculated with of AMF were significantly higher in most cases compared to noninoculated plants. The only exception to this general trend was observed in the case of P and K% as in the presence of AMF with control treatments they recorded insignificantly high er values compared to corresponding values in the absence of AFM. The obtained results are in agree ment with those obtained by various researchers that reported the potential effects of AMF on the accumulation of macronutrients in medicinal plant orangs (Chen and Zhao, 2009;Karagiannidis et al., 2011;AbdelKader et al., 2014;Vafadar et al., 2014;Oliveira et al., 2016;Yang et al., 2017;Moghith, 2019;Mohamed, 2020). The enhanced mineral absorption by AMF inocu lated plants could be elucidated by the efficiency of AMF to boost mineral affinities, reduce the critical concentration of elements absorption, augment the area of uptake and decrease the area of diffusion (Gashgaril et al., 2020).
Data in Table 6 also indicate that, in absence or presence of AMF foliar spraying with any concentra tions of PGRs resulted in significant increase in macronutrient in seeds in most cases, compared to control. With some exceptions recorded in absence of AMF as foliar application of CPPU resulted in insignificantly higher values of P% than the control, also in absence of AMF, foliar application of three tested PGRs resulted in insignificantly higher values of Ca % compared to control. Among PGRs, applica tion of NAA was the most effective treatment espe cially in presence of AMF. The results of increasing macronutrients accumulation due to NAA treatments are accordance with findings of previous studies (Alam et al., 2012;Atteya and El Gendy, 2018), while the obtained increase in macronutrients due to BA is similar to those described in numerous reports (Matter, 2016;Moussa, 2019;AbdelHamid, 2020;AbdelRahman and AbdelKader, 2020).

Content of micronutrients in seeds
As shown in figure 2 that under the same treat ments of PGRs plants inoculated with AMF had signif icantly higher values of Fe and Zn in their seeds than those grown in the absence of AMF. These results are in the same line of the findings of earlier authors (Chaudhary et al., 2008;Golubkina et al, 2020).
Data in figure 2 also showed that, in the absence or presence of AMF foliar application of any concen trations of PGRs resulted in significant increase in tested micronutrients (Fe and Zn) in seeds compared to control. The increases in the recorded values were more evident in the presence of AMF mostly with application of NAA. Such increase in Fe and Zn con tent due to BA treatments is in good agreement with those elicited by prior works (Baydar and Erdal, 2004;AbdelHamid, 2020).

Total protein
Data in Table 7 displayed that under the same PGRs treatments total protein percentage was signifi cantly higher in plants grown in the presence of AMF than those grown in the absence of AMF. These results are in conformity with that recorded by previ ous studies (Ouzounidou et al., 2015;AlAmri et al., 2016). Increasing protein due to AMF may be due to its role on inducing NH 4 + and NO 3 − absorption, and assimilation of these molecules into free amino acids that are involved in protein synthesis (Gashgaril et al., 2020).
The data in Table 7 also exhibited that in the absence or presence of AMF, spraying of plants with PGRs resulted in significant increase in total protein content compared to control. Among the tested PGRs NAA was superior in its effect predominately in the presence of AMF. The results of increasing pro tein content due to application of BA are in good agreement with those elicited by Prins et al., 2013, while such increase owing to CPPU treatments is coincided with those obtained by Abbas and Zahwan, 2016.

Total phenol content (TPC), Total flavonoid content (TFC) in in seeds
The data presented in Tables 7 showed that under the same treatments of PGRs TPC and TFC in seeds were higher in plants inoculated with AMF compared to noninoculated plants; however such increase was statically insignificant in the case of TPC compared to corresponding values in absence of AMF. The results of increasing TPC or TFC due to inoculation with AMF are analogy with that recorded by earlier workers (Amiri et al., 2017;Gashgaril et al., 2020).
Data outlined in Table 7 also indicate that in the absence or presence of AMF TPC and TFC in seeds were significantly higher in seeds of plants foliar sprayed with any concentrations of PGRs compared to control, with superiority of BA for increasing TPC and NAA for TFC especially in the presence of AMF. The present augmentations in TPC or TFC owing to NAA are in harmony with those obtained by previous author (Atteya and El Gendy, 2018), whereas increas ing in tested components due to BA are in the same line with the findings of earlier study (AbdelHamid,   2020; Sidkey, 2020).

Antioxidant activity in seeds
As shown from data listed in Table 7 that under the same treatments of PGRs antioxidant activity in seeds were higher in plants inoculated with AMF compared to corresponding values of noninoculated plants, however such increase was insignificant in most cases. The results of increasing antioxidant activity due to mycorrhizal treatments is supported by the results of other authors (Geneva et al., 2010;Amiri et al., 2017;Golubkina et al., 2020;Gashgaril et al., 2020).
Data recorded in Table 7 also indicate that in the absence or presence of AMF antioxidant activity was significantly higher in seeds of plants sprayed with PGRs concentrations compared to control, with supe riority of NAA especially in the presence of AMF, since recorded the highest values compared to con trol. The obtained increase in antioxidant activity due to NAA are in harmony with those obtained by previ ous author (Atteya and El Gendy, 2018), while the augmentation due to BA are in harmony with the finding of recent study (Sidkey, 2020).

Fixed oil percentage
It is obvious from data listed in figure 3 that under the same treatments of PGRs, the values of fixed oil % for plants grown in presence of AMF was significantly higher than corresponding values in the absence of AMF. The results of increasing fixed oil % owing to inoculation with AMF are in sequence with the find ings of earlier author (Moghith, 2019). Data in figure 3 also showed that, in the absence or presence of AMF in general application of PGRs resulted in significant increase in fixed oil % compared to control. These increases in the recorded values were more evident in the presence of AMF particular ly with application of NAA. Such augmentation in fixed oil due to application of BA is in agreement with those reported by previous study (Mousa et al., 2001).

Conclusions
Based on the outcome of the present research it could be concluded that, cultivation of chia plants in presence of mycorrhiza with foliar application of NAA at 50 ppm is recommended for enhancing growth, and nutritional values of seed yield. Fig. 3 Fixed oil % of Salvia hispanica as affected by the interac tion between plant growth regulators (PGRs) in absence () or presence (+) of Arbuscular mycorrhiza fungi (AMF) (mean of two season), column with different letters indi cate a significant difference at 5% level. Vertical bars indicate to standard error (SE) of three replicates.