Ameliorative effect of thiourea priming on germination characteristics of mungbean (Vigna radiata L.) under water and salinity stress

Drought and salinity are the major abiotic stresses that affect crop productivity particularly in arid and semiarid areas. Seed germination and seedling growth are the most critical stages that are adversely affected by stresses. Seed priming, an important technique to improve germination, involves exposure of seeds to an eliciting factor that hydrates the seed in a specific environment, followed by drying so that germination begins, but radicle emergence does not occur (Li et al., 2017 and Paparella et al., 2015). This makes the plants tolerant to future stress (Tanou et al., 2012). Under stress conditions, over accumulation of ROS causes the oxidative damage of cellular components, such as membrane lipids, pigments, enzymes and nucleic acids (Das et al., 2015 and Zavariyan et al., 2015). The stress tolerance can be improved with the exogenous use of stress alleviating chemicals (Farooq et al., 2009a, b). Among the stress alleviating chemicals, thiourea is an important molecule that can scavenge superoxide radicals or hydroxyl radicals (Singh and Rathore, 2003). The molecule of thiourea has two functional groups; ‘thiol’ that alleviates oxidative stress and ‘imino’ that partly fulfils the N requirement. Physiologically, thiourea offsets the effect of ABA and decreases the level of cytokinin in plant tissues subjected to water stress due to drought, salinity or supra optimal temperatures (Srivastava et al., 2010). Thiourea treatment effectively promotes germination when dormancy is related to salt or water stresses (Khan and Ungar, 2001). Being water soluble, readily absorbable in the tissues and capable to ameliorate stress effects; thiourea could be used to improve yield under abiotic stresses (Devi et al., 2015).
       
Mungbean [Vigna radiata (L.) Wilczek] is an important legume of dry land agriculture with rich source of proteins, vitamins and minerals. Capacity to restore soil fertility and short life span makes it valuable in various cropping systems particularly rice and wheat. India is the largest producer and consumer of mungbean and accounts for about 65% of the world acreage and 54% of the world production of this crop. It is the third most important pulse crop in India, occupying nearly 3.72 million ha area with 1.56 million tonnes production (Sehrawat et al., 2014). The remarkable research work has not been conducted under arid, semi-arid and rainfed conditions to find out the techniques for getting high germination percent, vigour index and productivity in mungbean.
       
Keeping above points in view the ameliorative role of thiourea under stress conditions and importance of mungbean crop in rainfed areas, the study was planned to investigate the extent to which thiourea priming can alleviate water and salinity induced stress on germination potential of mungbean.

The experimental study was carried out at Laboratories of the Department of Botany, Punjab Agricultural University, Ludhiana under ambient conditions during the month of March 2016 to assess the priming effects on germination parameters of mungbean seeds (cv SML 668). The seeds were unsoaked, hydroprimed (HP, distilled water) and thiourea primed at three concentrations viz. 750 ppm (TU1), 1000 ppm (TU2) and 1250 ppm (TU3). The seeds were germinated under simulated water stress with polyethylene glycol at osmotic potential of -0.2 and -0.4 MPa and salinity stress as NaCl at concentration of 30 and 50 mM. Healthy seeds of mungbean were air dried and stored at room temperature before treatment. The seeds were surface-sterilized by soaking in an aqueous solution of HgCl₂ (0.1%) for few seconds followed by two- three times rinses with distilled water. The seeds were blotted to dry and then used for germination experiments. Ten seeds were placed in Petri plates, lined with two layers of filter paper, for each treatment and each treatment was replicated thrice. For control, the filter papers were moistened with distilled water only. Petri plates were observed daily and remoistened as required. A seed with radicle length 2 mm was considered to be germinated. The experiment was observed for 7 days. After 3 days, the per cent germination was recorded and following parameters were calculated:
 
a) Per cent germination
The per cent germination was calculated on 3^rd day.

    

 
b) Seedling length (cm)
 
Seven days after germination the seedlings were removed from Petri dishes. The seedling length (cm) was measured with centimeter scale.
 
c) Seedling dry weight (mg)
 
The seven days old seedlings were kept at 70°C for 72 hrs in an oven followed by determination of their dry weight (mg).
 
d) Vigour index (VI)
 
Seedling vigour index was determined  as per formula given by Abdul-Baki and Anderson (1972).

       VI = Germination (%) × Total seedling length (cm)

 
e) Tolerance index
 
Tolerance index (TI) was determined as per the following formula given by Iqbal and Rahmati (1992).

   
The data on various parameters were subjected to statistical analysis (Singh et al., 2001). Critical difference values were calculated by doing analysis of variance (ANOVA) for factorial experiment replicated thrice in completely randomized design.

Thiourea primed seeds performed better in terms of per cent germination as compared to non-primed and hydro primed seeds under control as well as stressed conditions (Table 1). On an average the per cent germination declined with increasing water and salinity stress as compared to their respective controls but priming treatments enabled the seeds to cope with stress and retain significantly higher per cent germination than non-primed seeds. The non-primed seeds showed 34% decline in per cent germination under stressed conditions whereas the corresponding values were 24, 20, 20 and 25% respectively for HP, TU1, TU2 and TU3 seeds.
 

       
Seedling length increased with different thiourea priming treatments although the effect of different thiourea treatments was at par (Table 2). The growth of seedlings was adversely affected under stress conditions as depicted by a sharp decline in length under both water and salinity stress. The thiourea primed seeds have 12 per cent longer seedlings than hydro primed seeds under control. The seedlings from thiourea primed seeds were respectively 15 and 85 per cent longer respectively under -0.2 MPa PEG and -0.4 MPa PEG water stress than hydro primed seeds.  The seedlings from TU1, TU2 and TU3 treatments were at par with hydro primed under  salinity stress except TU3 treatment where seedlings were 29 per cent longer than hydro primed seeds under 50Mm NaCl. Thiourea priming enabled the seeds to cope with stress and retain significantly higher seedling length as thiourea primed seeds retained 47% of their respective control as compared to 28% retention by HP seeds under - 0.4 MPa PEG treatment.
 
  
 
Thiourea primed seeds have longer roots than hydro primed under all treatments indicating that thiourea promotes root system which is one of the most important physiological adaptation to cope with stressed conditions. Thiourea not only increased the root length but also increased branching in roots under simulated water stress. The ameliorative effect of thiourea on root length under water stress was more pronounced than salinity stress as depicted from data in Table 3. The root length of hydro primed seeds was at par with that of thiourea primed seeds under salinity stress.
 

       
Like root growth, shoot growth was also adversely affected under stress conditions as compared to control in all seedlings (Table 4). The shoot length of all seedlings germinated under control was longer than those germinated under different stressed conditions. Thiourea primed seeds performed better than hydroprimed under all conditions. The alleviatory effect of thiourea priming was more under water stress condition as compared to salinity stress.
 

       
The seedling growth in terms of seedling weight depicted that the growth of all seedlings was adversely affected under stress conditions (Table 5). All the seedlings growing under control accumulated the highest fresh weight 174mg that declined to 122 mg under stressed conditions (50 mM NaCl). On an average, the seedlings from thiourea primed seeds accumulated more weight than hydro primed seeds although the effect of different priming treatments was at par under stress conditions.
 

 
The root: shoot ratio represents the growth pattern of the seedlings. The higher root: shoot ratio indicates that the root growth is more pronounced (Table 6). The increase in root: shoot ratio represents the seedlings with pronounced root growth to have greater access to nutrients and water to support shoot growth under stressed conditions. The highest root: shoot ratio was observed in thiourea primed seeds under water stress (-0.4 MPa PEG) indicating that simulated water stress induced higher root growth to support the developing seedlings whereas under salinity stress no marked difference in the ratio was observed.
 
  
 
The seedling vigour was the highest for all the seedlings under control and declined under stressed conditions (Fig 1). The seedling vigour declined to least under salinity stress (50 mM NaCl). The vigour declined to 32 under salinity stress (50mM NaCl) for hydroprimed seeds whereas the corresponding value was 57 for TU3 seeds. The TU2 seeds retained higher seedling vigour i.e. 409 and 199 respectively under simulated water stress of -0.2 and -0.4 MPa PEG whereas the corresponding values were 204 and 83 for hydroprimed seeds. Thiourea primed seeds have higher seedling vigour than hydroprimed seeds under stressed conditions. 
 

       
The tolerance index represents the extent to which the primed seeds can cope with the stress and support their growth. Thiourea primed seeds have more tolerance index than thehydro primedseeds except under -0.2 MPa PEG where hydro primedseeds havemore tolerance than TU1(Fig 2). The tolerance index of thiourea primed seeds was more under water stress as compared to salinity stress indicating their higher ameliorative effect under water stress on germination and seedling growth of mungbean seeds as compared to salinity stress. This could be correlated to significant promotion of  root growth particularly branching in TU primed seeds under water stress in comparison to  salinity stress where the root growth was at par with control i.e. unstressed conditions.
 

       
Generally, water scarcity resulting from either drought or soil salinity influenced crop plant’s morphology, physiology and could lead to cellular and organeller deformation (Abdelkader et al., 2007 and Demirevska et al., 2009). The water and salt induced adverse effects on plants are mainly in the form of osmotic stress, specific ion toxicity, membrane damage, deficiency of essential nutrients, production of oxidants, etc. (Ashraf, 2009). Among the number of strategies used to alleviate these stresses, one is exogenous use of some potential organic and inorganic compounds as foliar spray or seed priming (Ashraf and Foolad, 2007). In our studies the per cent germination, seedling growth in terms of length and weight and seedling vigour declined in non-primed as well as hydro and thiourea primed seeds under induced water and salinity stress but the per cent decline in all the parameters reduced with thiourea priming. There are several reports concurrence with our results of decline in germination related parameters under stress conditions (Bethke et al., 2004 and Li et al., 2005). The reduction could be related to reduction in enzyme activities, retardation in the mobilization rate of soluble sugars (Ashraf et al., 2002) and effect on other metabolic and molecular responses (Ingram and Bartlets, 1996). Some earlier reports revealed that TU application improves stress tolerance and promotes germination (Yoshiyama et al., 1996) and enhances the yield of a broad range of crops, e.g., wheat (Sahu et al., 2006), mung bean (Mathur et al., 2006) and potato (Mani et al., 2012).
       
The ascribed TU-induced increase in growth could be due to triggering of a variety of physio-biochemical processes as recently reported by Pandey et al., (2013). The ameliorative effect of thiourea might be due to its potential to act as osmoregulator and its counteractive effect on ABA production (Kabar and Baltepe, 1989) that further control the adverse hormonal changes resulting from water stress induced by drought, salinity, or high temperature (Bano and Aziz, 2003). Further exogenous application of TU might have acted as a source of carbon and nitrogen that promoted growth of seedling (Mitoi et al., 2009 and Anjum et al., 2011).