Priming of Long-Term Stored Cotton Seeds Using Combined UV-A, B and C Radiation and Its Influence on Germination

The effects of ultraviolet (UV) radiation on terrestrial ecosystems and the agricultural sector have been extensively investigated due to the compelling issues such as climate change, thinning of the stratospheric ozone and enhanced level of solar ultraviolet-B radiation which can have serious impacts on global food security (Liu et al. , 2004; Van Dingenen et al. , 2009). The main three wavelength ranges of UV rays are UV-C (200-280 nm), UV-B (280-320 nm) and UV-A (320-400 nm). It was suggested that each 1 % reduction in ozone causes an increase of 1.3-1.8 % in UV-B radiation reaching the earth (Hollósy, 2002). Ozone is considered the only gas of the atmosphere that absorbs wavelengths shorter than 300 nm (Caldwell et al. , 1989). Therefore, most of the published research has focused on effects of elevated UV-B radiation on ecosystems and plants in particular.

Researchers have also investigated the potential benefits of radiation on plant growth induction, plant behavior and acclimation (Darras et al. , 2015; Qi et al., 2014; Singh and Datta, 2010). Since the discovery of ultraviolet waves, scientists have attempted to study the impact of these rays on seeds and plant growth (Krizek, 1975; Noble, 2002; Popp and Brown, 1933). UV irradiation of seeds produces free radicals which change cell membrane permeability and electric potential, presumably initiating diverse metabolic responses (Rogozhin et al., 2000). On the other hand, UV irradiation can affect DNA resulting in several photoproducts and pyrimidine dimers (Ravanat et al., 2001; Sancar and Sancar, 1988). It was reported that pre-sowing treatments of seeds using high energy radiation like laser, magnetic field and UV will eventually increase corps productivity by enhancing germination and seedling growth (Iqbal et al., 2016; Qiu et al., 2008; Thomas and Puthur, 2017).

Seeds priming by treating seeds with natural or synthetic compounds before sowing is performed so that seeds reach a physiological state where induction for stress tolerance and enhanced plant growth is achieved (Thomas and Puthur, 2017). Seeds priming enhances quality through the activation of pre-germinative metabolism including antioxidant functions and DNA repair mechanisms (Araujo Sde et al., 2016). Studies showed that plants grown from primed seeds exhibited higher tolerance against biotic andabiotic stresses (Borges et al., 2014; Iqbal et al., 2016; Mariz-Ponte et al., 2018; Ouhibi et al., 2014; Rashid et al., 2015; Zhang et al., 2016b). The aim of this work is to investigate the effect of pre-sowing UV-irradiation on germination and vigour under normal and stress conditions. This work will also study the expression of key growth and stress-related genes during the early stages of germination after UV treatment.

MATERIALS AND METHODS

UV irradiation platform setup

Seeds’ irradiation conditions

Four irradiation periods were chosen (4, 8, 12 and 16 min) and, at each exposure period, five seeds were irradiated together. The temperature and relative humidity in proximity to the irradiated seeds were registered. The ambient temperature and relative humidity of irradiated samples were maintained at 22°C ± 2 and 35 % ± 3 in order.

Plant material and cultivation

Plant material consisted of seeds of two Gossypium hirsutum L. accredited local variety Deir Al Zour 22 (a selected line from Delta Pine 41). It is noted, through repeated trials and experience that seeds vigour decrease by time of storage. Germination in the field reached 30 % using two years old seeds. Seeds were provided by the Cotton Research Administration (CRA) and were stored at 4 °C for five years. In pots experiments, irradiated cotton seeds were sown (5 seeds) in pots (7 X 9 cm) which contain universal potting soil (3 pots per treatment). Pots were kept in a growth cabinet under 16 hrs light and a temperature of ~ 25 °C. In vitro culture of irradiated cotton seeds was conducted using Murashige and Skoog (MS) media supplemented with MS vitamins (4.4 g/L), agar (9 g/L), and sucrose (30 g/L). Testing the effects of both salt and drought stress on irradiated and control cotton seeds were performed in vitro at 25 ºC and 16 hrs light. The salt treatment included 75, 125 and 175 mM NaCl and the drought treatment included the application of PEG 6000 to generate osmotic pressure of -0.2, - 0.5 and -1 bar. Fifteen tubes (one seed per tube) were used for each treatment.

Flow Cytometry

The method described by Jin et al. (2008), was used to analyze nuclear DNA content to determine the variations in cell cycle due to UV treatments (Jin et al.,

2008). Samples used for flow cytometry were dry and non-irradiated cotton seeds, dry and irradiated (4, 8, 12, and 16 min) cotton seeds, 2 DAP (days after planting) roots from non-irradiated cotton seeds, 2 DAP roots from irradiated (4, 8, 12, and 16 min) cotton seeds, 6 DAP roots from non-irradiated cotton seeds, 6 DAP roots from irradiated (4, 8, 12, and 16 min) cotton seeds.

Plant tissues (seeds, and roots) were rinsed with 2% filtered bleach and washed thoroughly with sterile filtered deionized water. The plant cells nuclei in those tissues were mechanically extracted by chopping with scalpel in the presence of Ice-cold extraction buffer (70 mM NaCl, 0.2 mM EDTA-acetic acid, 0.1M Tris, 0.5% (v/ v) Tween20, pH 7.5). For each sample, nuclei preparation was filtered through a 50-µm nylon mesh, followed by centrifugation at 500 xg (for 10 min, at 4°C). Then the supernatant was discarded and cell nuclei were suspended in 1 ml extraction buffer containing propidium iodide (50µg/ml). This preparation was then analyzed on BD Biosciences FACSCaliburTM flow cytometer (BD Biosciences, USA) with doublet discrimination module on. At least 10,000 nuclei were analyzed per sample. Three samples (replicates) for each treatment were analyzed. Flow cytometric data acquisition was performed using CellQuest software (BD Biosciences, USA). Cell cycle data analysis was done using ModFitLT V3.2 software (Verity Software House, USA).

Gene expression analysis

Gene expression analysis of each of VPP , DEH and GA3ox1 genes was conducted using cDNA synthesized from RNA of the following samples: UV non-treated cotton seeds in three stages (dry, 1, and 3 DAP), and 12 min UV irradiated seeds in three stages (dry, 1, and 3 DAP). Seeds were planted in wet potting compost in potting trays and were irrigated daily.

Total RNA was isolated following a modified and rapid isolation protocol (Jawdat and Karajoli, 2012) and DNA contamination was eliminated using the TURBO DNA-freeTM (Ambion/life technologies, USA) following the manufacturer’s manual. The iScriptTMSelect cDNA Synthesis kit (Bio-Rad, USA) was applied to produce, cDNA starting from 1 µg RNA per sample. Real-Time PCR was performed in two replicates in a 25 µl of a reaction mixture composed of 12.5 µl IQ SYBR super mix (BioRad, USA), 1 µl of cDNA, 1 µl of each of the forward and reverse primer (10 µM), and 9.5 µl of d.d. water. The quantification of mRNA levels was normalized with the level of mRNA for GhEF1α5 (Artico et al., 2010). The relative expression (fold expression) was calculated using the ـ∆∆Ct method as follows:

2-∆∆Ct = 2 ˗ ((Ct treated target gene– Ct treated reference gene) ˗ (Ct untreated target gene–

Ct untreated                ))