Comparative Effect of Seed Priming and Growing Media on Germination and Seedling Rootstocks of Mango

The poor seedling rootstock is a key factor in mango yield reduction. Container-based study was conducted to evaluate the effect of seed priming and growing media on seedling rootstocks of mango. Hydropriming of seed was performed by using distilled water, while gibberellic acid (GA3) and NPK fertilizer (Solo plant) were used for hormonal and nutripriming, respectively. In second part, the primed and unprimed seeds were planted in four different growing media (GM) including GM1, GM2, GM3 and GM4. Data showed that seed germination (%), germination index (GI), seedling vigor index (SVI), height of seedling, stem diameter, chlorophyll content, electrolyte leakage of leaf, nutrient (N, P, K, Ca and Mg) contents in leaf tissue was significantly altered by both seed priming and growing media respectively. In case of priming treatments, hormonal primed seeds had the best seed germination (77.01%), stem diameter (9.65 mm) and electrolyte leakage of leaf (13.01%); while N (1.13%), P (0.14%), K (0.87%), Ca (2.40%) and Mg (0.34%) content of leaf tissue was observed maximum in nutripriming treatment. Whereas, seed germination (71.96%), germination index (11.15) and stem diameter (9.41 mm) had greater values in response to the GM2 treatment. While GM4 grown seedlings had higher seeding height (33.81 cm), N (1.17%), P (0.15%), K (0.98%), Ca (2.64%) and Mg (0.38%) content of leaf tissue. It is concluded that seed germination and seedling growth attributes had a greater influence of GA3, while mineral nutrient contents of leaf had a significant effect on nutripriming. Among growing media, GM2 was observed better for growth parameters while GM4 for mineral nutrient contents of leaf..


Introduction
Mango is generally propagated by sexual and asexual methods Pinto et al., 2018). There are two distinct types of mango varieties including monoembryonic and polyembryonic. Seedling that is raised from monoembryonic seeds is not considered as true-to-type mango. Generally, both zygotic and nucellar seedlings may be used as a rootstock (Ballyise, 2006;Kolekar et al., 2017). Typically, when mango plants are raised by seed, they lose their many unique features, consequently, vegetative propagation methods including grafting and budding become important to preserve and perpetuate the characteristics of each cultivar of mango (Abbas et al., 2015). Healthy seedling rootstock is the main basis for successful and sustainable fruit production of mango in Pakistan (Kumar et al., 2008). Seed priming technique and growing medium are considered as main starters for the seed germination and growth of the seedlings (Aklibasinda et al., 2011;Aatlai and Srihari, 2013;Pinto et al., 2008). Mango seeds have poor viability due its recalcitrant nature. According to (Gill et al., 1985) the viability of the mango seeds is typically declined after 15-days of the fruit harvest. Mango seed initiates to germinate about twelve days after planting and may finish within thirty days of planting (Thakriya et al., 2017). The delay in mango seed germination is typically due to its hard seed coat which is impermeable for water and gases and either due to deficiency or excess of growth hormones (Basra et al., 2005). Seed priming is recognized as an effective and efficient method to improve seed germination potential and seedlings emergence (Shehzad et al., 2012;Ramírez and Davenport, 2010). Usually, old mango seeds have low viability than fresh seeds. Storage of seeds may also reduce the germination potential (Khan et al., 2006). Thus, seed priming can increase germination potential of mango seeds. Considering seed morphology and physiology, various seed priming methods have been formulated for improving its germination and emergence potential such as (a) hydropriming in which water used for soaking of seeds, (b) osmopriming where solutions having different osmotica are used for seed soaking, (c) halo priming in which seed is soaked with salt solution, (d) thermo-priming in which seeds of crop is treated under low or high temperatures extremes, (e) bio www.joarps.org priming, when seed is treated with inoculum and (f) nutripriming in which seed is treated in essential plant nutrients solution. Seed priming has been used commercially to enhance seed germination potential of several crops including field as well as fruit crops. However, this technique is less utilized to improve seed germination potential of mango crop. Another driving factor which plays key role in sustainable production of mango is use of growing media for seedlings establishment. The properties of growing media such as nutrients availability, water holding and cation exchange capacities are closely linked to its content of organic matter. Further, these media may also facilitate in root penetration and provide better aeration consequently improve mango rootstock seedlings (Supriya and Polara, 2015;Mhango et al., 2008). A wide variety of materials are being used and mixed in different ratios for formulation of growing media for the establishment of rootstock seedlings of mango. Those include organic materials such as peat, sawdust, coconut husk, sugar mills industrial waste, etc. and inorganic materials such as sand, canal silt, perliteetc. (Donovan et al.,2016;Ryan et al., 2001). The composition and formulation of each growing medium may depend on type of crop, locality and its availability (Ryan et al., 2001). Many commercial growing media are available in the market of advanced countries for raising and establishing healthy seedlings of different crops. However, commercial media availability is a big hurdle for raising mango rootstock seedlings in under developed countries such as in Pakistan. Hence, this study was designed to formulate low-cost but having high nutrients availability growing media for mango rootstock seedlings.

Materials and Methods
Seed priming technique: The mango fruits of Sindhri cultivar were collected from the commercial orchards. The seeds were extracted and air-dried ( Figure 1).

Figure 1. Drying of the mango seeds
The seeds were primed in various priming solutions and unprimed seeds were treated as control. The distilled water was used for hydropriming. Hormonal priming was performed by using gibberellic acid @ 100 mg L -1 (Sigma Aldrich). The NPK fertilizer (solo plant) @ 100 mg L -1 was used for nutripriming. The process of seed priming was conducted for 48 hours. The primed seeds were planted in polythene bags contained various growing media combination as described below. Formulation of growing media: To prepare following growing media (GM) combinations, cheap materials available in the vicinity of Tandojam such as orchard soil, canal silt, bagasse and press mud from sugar mill industry were collected from local sugar mill to formulate following GMs (Figure 2 & 3). GM1: Orchard soil (farmer's practice) GM2: 70% Bagasse + 5% Coco peat + 25% Canal silt GM3: 60% Bagasse + 5% Coco peat + 35% Canal silt* GM4: 20% Bagasse + 20% Coco peat + 30% Canal silt + 30% Press mud *GM3-Standard practice developed under ASLP (Australian Sector Linkage Program) project at Mango Research Station Shujabad. Soil analysis:The composited soil and canal silt samples were collected brought in the laboratory, air-dried and ground by using 2 mm sieve. The electrical conductivity (EC) and pH of the soil-water extract (1:5) were measured with pH and EC meters respectively. Walkley-Black method was used for the measurement of organic matter (OM) content. The total Nitrogen using Kjeldahl's distillation, whereas Olsen method was used for determination of available Phosphorus. Flame photometric technique was used for the measurement of exchangeable K, Ca and Mg (Bremner, 1965). Analysis for growth media components: The pH and EC of the bagasse, cocopeat and press mud were measured using saturated media extract (SME) method followed by pH and EC meters were used. Total Nitrogen of bagasse, cocopeat and press mud was analyzed by Kjeldahl's method (Richards, 1954). The P (Estefan et al., 2013), K, Ca and Mg contents were measured by using atomic absorption spectrophotometer. Leaf tissue analysis: Fully matured leaves were collected from grown seedlings. The leaves were brought to laboratory, washed; oven dried (68ºC) and wetdigested in mixture of perchloric (HClO4) and nitric acid (HNO3) at the ratio of 1:5 and filtered. The filtrate was used for analysis of Phosphorus by using vanadomolybdo-phosphoric acid method (Cottenie, 1980) whereas flame photometer was used for analysis of potash (Knudsen et al., 1982). The EDTA method was used for the determination of Ca and Mg (Knudsen et al., 1982). Plant data: The plant data was recorded for seed germination (%) (GP) mean germination time (MGT), germination index (GI), seedling vigor index (SVI), height of seedlings, stem diameter and chlorophyll contents. Seed germination was recorded for one month at interval of one week. The seed germination (%) was computed by following formula (Larsen and Andreasen, 2004). The five random seedlings of each treatment were selected for seedlings height. The seedlings height was measured from tip to the base of the plant at 30 days interval. Stem diameter (mm) was determined by using a digital vernier caliper at three points (center, top, and bottom of the stem). Chlorophyll content was determined by using SPAD 502. Statistical analysis: The entire data so collected were statistically analyzed by using Statistix Software package (Ver. 8.1). The effects of the main factors and their interaction with each other were determined. Experiment was laid out according in completely randomized design factorial. The superiority of the treatments was evaluated by applying Least Significant Difference test at p<0.05.

Results
The means of the seed priming depicted greater variation in results of the seed germination percentage, Germination index, and Seed vigor index (Table 1). Each seed priming treatment had a germination percentage of more than 50% except nutriprimed seeds which had germination of 48.61%, even lower than control (68.81%). The greatest mean for seed germination (77.01%), germination index (10.82) and seedling vigor index (4271.75) was observed in hormonal primed seeds. However, germination index was also similar to the results obtained from hydro (10.71) and nutriprimed (11.08) seeds. The SVI was also found greater in unprimed seeds (2490.60) as compared to nutriprimed seeds (2151.37). In case of growing media effect (Table  1), seed germination was observed more than 50% from each growing media producing the greatest (71.96%) in GM2. However, the germination index (11.15; 10.95) and SVI (3147.87; 3170.72) was observed similar to GM2 and GM4, respectively. The minimum seed germination (57.84 %), germination index (8.80) and SVI (2550.90) noticed in control treatment. In case of interaction between seed priming and growing media, only SVI produced significantly the greatest seedling vigor index (4875.1) in hormonal seed priming and GM2. Further, similar response (4436.4) was also observed in hormonal seed priming and GM4 treatment. The seedling height and stem diameter were significantly altered by seed priming and growing media treatments (Table 2). However, interaction of both seed priming and growing media treatments had no effect on seedling height and stem diameter of mango seedlings. The mean seedling height ranged from 23.15 (control) to 35.45 cm (hormonal priming). Hormonal seed priming also produced greater stem diameter (9.65 mm) than any other primed treatment. Moreover, seedling height and stem diameter in hormonal priming was also found significantly different from nutripriming (33.28 cm; 8.59 mm) and hydropriming (29.30 cm; 8.16 mm) treatments. In case of growing media (Table 2), seedlings height followed an order of GM4> GM>GM2>GM1. Whereas, stem diameter of rootstock seedlings planted in different growing media followed a different growth trend. Stem diameter of seedlings ranged from 7.72 mm (GM4) to 9.41 mm (GM2). The mean chlorophyll contents and electrolyte leakage significantly differed in seed priming treatments ( Table  3). The interaction of seed priming and growing media effect was only significant on leakage of the electrolytes from leaf. The hormonal primed seeds produced seedlings with higher mean chlorophyll content (53.76rg) followed by nutripriming (51.14 rg). Unprimed seeds produced seedlings with lower chlorophyll content (35.64 rg) and with more leakage of the electrolytes (16.10%). The effect of growing media on chlorophyll content indicated that GM2(47.88 rg) and GM3(49.31 rg) grown seedlings were statistically similarthan GM1 (42.51 rg) and GM4 (45.50 rg). The interaction effect of seed priming and growing media revealed that unprimed seedlings had greater leakage of the electrolytes (17.23%) than GM3. The N content of leaf was significantly differed by both seed priming and growing media ( Table 4). The interaction of seed priming and growing media found non-significant. The means of the seed priming depicted that N content of leaf was more than 1% from each priming treatment withmaximum (1.13%) from the seedlings grown in nutripriming. The seedlings from unprimed (1.03%) and hydroprimed (1.04%) seeds had a similar N content of the leaf. On the basis of growing media, the mean N content of GM1 was less than 1% i.e. 0.92%. The GM4 grown seedlings had more N content (1.17%) following by GM2 (1.08%). and K (0.79; 0.80%). While unprimed grown seedlings had less P (0.12%) and K (0.71%). The maximum P (0.14%) and K (0.87%) content of leaf were observed in nutripriming. The GM4 grown seedlings had maximum leaf P (0.15%) and K (0.98%). The GM2 and GM3 had similar leaf content of P (0.13; 0.13%) and K (0.90; 0.89%). The Ca and Mg content of leaf were also significantly affected by seed priming and growing media and their interaction ( Table 6). The interactive effect of seed priming with each growing media depicted that seedlings had Ca content of more than 2% in each treatment of the interaction except the interaction of GM1and unprimed seeds. The Ca content of GM1 grown seedlings range from 1.20 to 1.60% with a mean value of 1.44%.
However, seedlings raised in GM2, GM3 and GM4 media depicted similar results of CaandMgin response to the unprimed or hydro or hormonal primed seeds. Only seedlings grown from nutriprimed seeds produced better results in each growing medium. Seedlings had maximum mean Ca (2.40%) and Mg (0.34%) content in nutripriming; while GM4 had better mean results for Ca (2.64%) and Mg (0.38%).

Discussion
Seed priming is a widely accepted method to achieve uniform germination and high-quality seedlings of crops (Ma et al., 2018) In view, different seed priming techniques were compared to observe the response of mango rootstock seedlings to hormonal, nutripriming and hydropriming techniques. The results of present study indicated that hormonal priming showed promising results for enhancing seed germination percentage, germination index, seedling vigor index, seedling height, stem diameter, chlorophyll content and electrolyte leakage of the leaf than rest of the priming techniques. This might be due to gibberellic acid (GA3) role in the stimulation of amylase synthesis and production that could hydrolase starch into endosperm and provide sugars, consequently encouraged seed germination (Matilla et al., 2008;Voegel et al., 2011). According to Kolekar et al., (2017) the application of GA3 (@100 ppm) could increase seed germination and SVI of mango. Similarly, Venkat and Reddy (2005) and Shaban (2010) indicated that 100 or 200 ppm amount of GA3could be beneficial for improving seed germination of rootstock seedlings of mango. In some other studies, it has been indicated that greater rates (i.e.500 to 1000 ppm) of GA3 may be considered to improve seed germination of rootstock seedlings of mango . Further, GA3 also increased plant height, stem girth and number of leaves of mango as in this study. This was likely due to alteration in meristematic tissues of mango (Venkat et al., 2006;El-Zaher, 2008). Further, response of GA3 may also be differed in varieties of mango due to differentiation in development of meristematic tissues of mango (Mobli and Baninasab, 2008). The increase in chlorophyll content of leaves in GA3primed rootstock seedlings as compared to other seed priming techniques in present study was attributed due to increased rate of photosynthesis. Further, the increased chlorophyll content was likely due to involvement of growth hormones in the synthesis of chlorophyll molecule (Kanjilal et al., 1998;Shah, 2007). Some studies also showed that the synthesis of chlorophyll content of rootstock seedlings of mango was also affected by the rate of GA3 (Mostafa and Alhamd, 2011;Jayantilal, 2015).
The production of rootstock of superior quality is the pre-requisite for plantation of a mango orchard (Mngombaet al., 2010;Kaur, 2017). In this study, various growing media were compared to formulate low cost and good quality growing media for rootstock seedlings of mango. Among all tested growing media, GM2 produced better seed germination (%), GI, SVI, stem diameter and chlorophyll content. These results were likely due to increased aeration and drainage properties by coco peat and bagasse materials (Sarkar et al., 2005). Generally, bagasse and coco peat may improve physical and chemical properties of growing media and resultantly improved seedlings development in nursery (Abad et al.,2002;Basirat, 2011). In recent study (UlHaq et al., 2017) indicated greater mango rootstock seedling survival in growing medium having bagasse (70%) as major ingredient. In contrast, Memon et al. (2017) observed non-significant differences in growth parameters of mango rootstock in media containing low amount of bagasse in mixture.
The nutrient content is also one of the key criteria for selection of growing media for nursery development. In present study, leaf N content in seedling of mango rootstock was found in the range of the critical levels mentioned in Table 7 (Young and Koo, 1971;Samra et al., 1978;Catchpole and Bally, 1995;Robinson et al., 1997;Poffley and Owens, 2005). Further, P and K levels were also found in the established ranges of mango seedlings. However, Ca content was noticed low as compared to previously reported levels (Young and Koo 1971;Young, and Sauls, 1981) while level of Mg found satisfactory. Further, these typical differences in critical levels of nutrients might be linked to combination of rootstock and scion, time of sampling, age of plant, propagation mode and varieties of mango (Ryan et al.,2001;Zuazo et al., 2006).

Conclusion
It was concluded that seed germination (%) and seedling growth attributes of mango had a greater influence of gibberellic acid seed priming, while mineral nutrients content of leaf was significantly affected by nutripriming than any other seed priming technique. Among growing media, GM2 was observed better for growth parameters while GM4 for mineral nutrients content of leaf.

Competing Interests Disclaimer
Authors have declared that they have no known competing financial interests or non-financial interests or personal relationships that could have appeared to influence the work reported in this paper.