Plant material

Saffron corms were obtained from Bloembollenbedrijf J.C.Koot (Vennewatersweg, The Netherlands). We peeled the corms (Fig. 1A) and discarded those injured or infected. Next, we dipped the corms in a fungicide solution that contained 75 mg·L⁻¹ metalaxyl and 175 mg·L⁻¹ copper oxychloride (Aromil-Plus 50 WP; Mobedco-Vet, Amman, Jordan) to prevent fungal infestation and dried them before planting. We planted corms into plastic pots (6 cm diameter) filled with a mixture of perlite and peatmoss (1:1; v/v) to initiate sprouting. The pots were irrigated and incubated at 14 °C in the dark in a controlled temperature-growth chamber for 1 week followed by incubation at 12 °C for another week.

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Growing substrates and mode of nutrient supply in hydroponics

We transplanted sprouted corms [3–5 cm length (Fig. 1A–C)] of the size 4 (10–11 cm circumference, 3.2–3.5 cm diameter) into hydroponic plastic pots (10 cm × 8 cm) filled with either volcanic rock or perlite, and placed the pots onto floating polyurethane foam board (60 cm × 40 cm × 3 cm) purchased from a local supermarket. Each board had 20 pots and was placed in a tank to supply the plants with a nutrient solution (Nabtah, HydroArabia; Hasad Al-Dahab Factory for Liquid Fertilizers, Riyadh, Saudi Arabia). We adjusted the electrical conductivity (EC) of the nutrient solution to 1.4 dS·m⁻¹ using a multiparameter bench meter (MI 180-US; Milwaukee Instruments, Rocky Mount, NC) and maintained it by providing a fresh solution with the pH value adjusted to 5.8 ± 0.2. The nutrient solution contained (mg·L⁻¹) 163.20 nitrogen (N), 34.53 phosphorus (P), 172.56 potassium (K), 105.11 calcium (Ca), 33.83 magnesium (Mg), and 62.70 sulfur (S) as macronutrients. Micronutrients of the solution were composed of (mg·L⁻¹) 1.83 iron (Fe), 0.23 boron (B), 0.27 manganese (Mn), 0.19 zinc (Zn), 0.12 copper (Cu), and 0.07 molybdenum (Mo). Saffron corms were grown in either the ebb-and-flow system or the continuous immersion system. A layout of the hydroponic systems used in this study is presented in Fig. 2. We programmed the ebb-and-flow system to immerse the plants in the nutrient solution for 6 or 12 h (six times per day with varied immersion durations of either 1 or 2 h in each cycle). For the continuous immersion system, air of 0.3 v/v/min (air volume/nutrient solution volume/minute) was supplied into the nutrient solution. The plantlets were continuously submerged in the solution to maintain a constant nutrient and water status in the root zone. We exposed sprouted corms to a gradual air temperature decrease for 2 weeks, starting from 10 °C and finishing with 8 °C (2 °C per week) and subsequently cultivated them at 8 ± 1 °C until harvesting daughter corms at the leaf senescence stage. Light intensity was adjusted to 100 μmol·m⁻²·s⁻¹ photosynthetic photon flux density for a 16/8 h light/dark cycle provided by cool white fluorescent lamps.

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Growth conditions of corms of different sizes in hydroponics

We graded saffron corms (Fig. 3) into four groups as follows: size 1 (7–8 cm circumference, 2.2–2.5 cm diameter), size 2 (8–9 cm circumference, 2.5–2.8 cm diameter), size 3 (9–10 cm circumference, 2.8–3.2 cm diameter), and size 4 (10–11 cm circumference, 3.2–3.5 cm diameter). We cultivated all of the corms in a volcanic rock substrate in the continuous immersion system with an air supply of 0.3 v/v/min to evaluate their flowering, growth performance, and cormlet production parameters. All culture conditions were set as described previously.

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Flowering, vegetative growth, and cormlet formation parameters

We recorded flowering parameters (number of flowers per plant, stigma length, and stigma fresh and dry weight) 29 d after corm transplantation and during the flowering period of 20 d. Stigma dry weight was determined after drying for 1 h at 40 °C (Maggio et al., 2006). We also measured vegetative growth parameters (plant height, fresh and dry weight of leaf, and leaf area) 22 weeks after corm sprouting. Leaf dry weight was determined after oven drying for 48 h at 70 °C. After leaf senescence, we pulled out the plants, removed mother corm parts, and separated replacement cormlets. Cormlet formation parameters from 15 randomly selected plants (number of cormlets per plant, cormlet diameter, and cormlet fresh weight) was recorded 24 weeks after corm sprouting.

Photosynthetic parameters

We recorded photosynthetic rate, and g_S using a portable photosynthesis system (LI-6400; LI-COR Biosciences, Lincoln, NE) equipped with a standard 2 cm × 3 cm leaf cuvette and a light source (LI-6400-02B; LI-COR Biosciences). We measured the parameters under the carbon dioxide (CO₂) concentration in the inflow air of 350 μmol·mol⁻¹ under the relative humidity values of 60% and leaf temperature of 8 °C at ambient conditions. We performed the measurements on the young leaf of three randomly selected plants from each treatment.

Experimental design and data analysis

The experiment on growing substrates and mode of nutrient supply was set up using a completely randomized factorial design, whereas the experiment on different corm sizes was set up using a completely randomized design. The factorial design has two substrates (volcanic rock and perlite) and two modes of nutrient supply (ebb-and-flow system and aerated continuous immersion system). Each treatment had three replicates and each replicate was represented by a floating tray that contained 20 pots; each pot contained one plant. We assessed the effects of the treatments using analysis of variance and Tukey’s multiple range test (SAS ver. 6.12; SAS Institute, Cary, NC).

Effects of growing substrates and the mode of nutrient supply

Both the growing substrate type and the mode of nutrient supply to the roots of saffron plants, either by the continuous immersion or the ebb-and-flow system, significantly influenced vegetative growth, photosynthetic capacity, and cormlet production (Table 1). Saffron plants grown in the volcanic rock–based continuous immersion system displayed the highest plant height, leaf fresh and dry weight, and leaf area compared with other treatments. The continuous immersion system was better for saffron growth than the ebb-and-flow system. In the ebb-and-flow system, immersion of saffron roots for 6 h showed better results than the 12-h treatment that rendered the lowest growth parameters and cormlet production values. The highest number of cormlets (5.8), average cormlet diameter (24.1 mm), and weight (6.62 g) were recorded from the volcanic rock–based continuous immersion system. Conversely, we did not find a dependence of saffron flowering parameters on the type of hydroponic system, growing substrate type, or their interaction, except for the stigma length that was significantly influenced by the hydroponic system type (Table 1). Stigma length was the highest (41.8 mm) in the continuous immersion system.

Table 1.Flowering, growth characteristics and cormlets production of saffron [size 4 (3.2 −3.5 cm diameter)] as affected by different hydroponic culture system and growing substrates in a controlled-environment growth chamber.

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It has been reported that temperature, among several environmental parameters, plays a pivotal role in saffron flower induction (Kafi et al., 2002; Molina et al., 2005a, 2005b). Therefore, saffron corms were incubated at a moderately high temperature (23 to 27 °C) to induce flowering, followed by the exposure to a moderately low temperature (17 °C) for floral emergence. Under field conditions, floral initiation usually occurs during summer, coinciding with the revival of meristematic activity in the apical bud. Thus, corms can be directly cultivated in the field to force flowering or can be stored up to 60 d at 2 °C to extend and facilitate harvesting (Amooaghaie, 2007; Mollafilabi et al., 2017). Amooaghaie (2007) reported that storing saffron corms at a freezing temperature damaged them, and prolonged cold storage delayed flowering and greatly reduced stigma yield. In the present study, saffron corms were exposed to a gradual air temperature decrease for 4 weeks, starting at 14 °C and finishing at 8 °C (2 °C per week). First emergence of flowers was observed 29 d after corm transplantation (Fig. 1D and E), and harvesting of flowers (Fig. 1F and G) lasted for 20 d. Earlier flowering in a growth chamber compared with glasshouse-grown plants (days 93 vs. 103 after transplanting), was reported by Maggio et al. (2006).

Selection of suitable substrates and setting the optimal environmental control can significantly affect plant growth (Dewir et al., 2004, 2005a, 2005b; Saleh et al., 2019). We found that the type of growing substrates (perlite and volcanic rock) had no significant effect on flowering traits, but significantly influenced plant growth, photosynthetic rate, g_S, and cormlet formation (Table 1, Fig. 1H). Maggio et al. (2006) reported similar observations for corms forced in a cold glasshouse, in which perlite and vermiculite substrates did not affect flowering yield, but only a larger leaf area per plant was obtained in vermiculite in comparison with perlite.

In the present study, root formation of saffron plants was affected by the mode of nutrient supply and growing substrate type. Contractile roots, short, and thick fibrous roots were observed in a perlite substrate (Fig. 1I), whereas thinner and longer ones were observed in volcanic rock (Fig. 1J). The aerated continuous immersion system favored growth of fibrous roots compared with the ebb-and-flow system. A previous study by Souret and Weathers (2000) indicated that saffron roots were shorter, thicker, and contractile and that their growth was significantly reduced in a soilless culture compared with culturing in soil. Previous studies offered explanations of this phenomenon, including mechanical resistance that impedes root growth (Zobel, 1991), presence of light conditions that suppress root growth (Poovaiah and Reddy, 1991; Souret and Weathers, 2000), or oxygen deficiency that leads to hypoxia and alters root growth. However, intensive root growth of saffron plants observed in the applied continuous immersion system might be promoted by continuous aeration providing sufficient oxygen to the roots, accounting for vigorous plant growth and increased cormlet formation compared with the ebb-and-flow system.

Effect of corm size on growth, photosynthetic, and yield parameters

Table 2 presents the effect of corm size on flowering, growth, and cormlet production of saffron grown in the aerated continuous immersion hydroponic system. Corms that were small [size 1 (2.2–2.5 cm diameter)] did not flower. Flowering parameters (number of flowers, stigma length, stigma fresh and dry weight) increased with increased corm size. Similarly, vegetative growth and photosynthetic parameters, as well as cormlet production, were affected by corm size. Corm size 4 (3.2–3.5 cm) recorded the highest values of flowering and vegetative growth parameters, as well as of cormlet production (Table 2). Saffron has a globular corm with diameters ranging from 0.5 to 5.0 cm, and its weight ranges from 1 to 20 g, depending on environmental conditions and agricultural management (Ali et al., 2018; Douglas et al., 2014). Each corm can produce one to four apical shoots containing developing flower and leaf (Ali et al., 2018). Mother corm size and weight have a significant impact on flowering and the number of daughter corms produced (Ali et al., 2018; Bayat et al., 2016; Douglas et al., 2014). Small mother corms (<1 cm diameter and <6 g fresh weight) do not flower and produce nonflowering cormlets (Ali et al., 2018; Douglas et al., 2014). Previous reports indicate that large corms (≥10 g) produce a high yield of stigma and numerous cormlets because they serve as a reservoir of nutrients and have a greater ability to take up water and nutrients than small corms (Ali et al., 2018; Douglas et al., 2014; Ghobadi et al., 2015; Koocheki et al., 2016a, 2016b; Mollafilabi et al., 2013). By decreasing corm weight, stigma yield and daughter corm production drop significantly (Askari-Khorasgani and Pessarakli, 2019; Koocheki et al., 2016a). From the commercial point of view, a corm that is 2.5 to 3.5 cm in diameter (8–16 g and mostly 8–11 g) appears to be the most common corm size used for planting (Douglas et al., 2014; Koocheki et al., 2016a). In a hydroponic system, corm size plays a critical role in saffron flowering. It was observed that only corms >2.6 cm diameter bloomed (Souret and Weathers, 2000). In addition, small corms (<2.6 cm) presented poor flowering in aeroponics, hydroponics, and soil (Souret and Weathers, 2000). Cavusoglu and Erkel (2005) studied the effect of size of greenhouse-cultivated saffron corms and found that the number of flowers and time of flowering were both dependent on the size of mother corms. Corm size plays a central role in the life cycle of saffron because a corm provides storage for photosynthetic products that are most needed after dormancy and at early stages of crop growth (Alvarez-Orti et al., 2004). Corm size has also been shown to affect the production of saffron flowers (De-Maastro and Ruta, 1993) and differences in the period required for flower initiation have been attributed mostly to the corm size (Negbi, 1999). In larger corms, cell division and, consequently, leaf growth occur somewhat earlier than in smaller ones. Therefore, saffron plants obtained from larger corms produce more leaf area compared with those from smaller corms (De-Maastro and Ruta, 1993). Earlier growth and larger leaf positively affect the photosynthetate accumulation, finally resulting in larger daughter corms at the end of the growing season (Molina et al., 2005b).

Table 2.Performance of flowering, growth characteristics, and cormlets production of saffron grown in volcanic rock substrate as affected by corm size in the continuous immersion with air supply hydroponic culture system in a controlled-environment growth chamber.

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