Seed Hydropriming Duration for Potential Seedling Vigour in Pigeon Pea [Cajanus cajan (L.) Millsp.], Sunflower (Helianthus annuus L.) and Rice (Oryza sativa L.)

In the climate change scenario, drought situation is the predominant threat to crop production under rainfed conditions (Ocvirk et al., 2020) and drought is generally accompanied by heat (Krishna et al., 2021) leading to more than a 50% reduction in the yield of crops (Bukhari et al., 2019). The primary constraints for productivity under rainfed conditions are poor seed germination, seedling emergence and crop stand (Singh et al., 2015; Finch-Savage and Bassel, 2016; Fahad et al., 2017). The seedling stage is more sensitive to drought stress (Nanja Reddy et al., 2021). Hydropriming or seed hardening is one of the approaches to improve seed performance and offset the negative effects of abiotic stresses (Rajashekara et al., 1970; Hussain et al., 2017). Hydropriming is the rapid uptake of water that induces partial hydration under in vitro conditions to initiate the germination process but not the protuberance of radical (Fahad et al., 2017). The priming was reported to enhance the hydrolytic enzymes like alpha-amylase activity to increase the conversion of starch to glucose for the respiration process of seed germination in cereals (Zhao et al., 2018; Ren et al., 2023), proteases (pulses) and lipases (oil seeds). Hydropriming improves the metabolism and transport of proteins, free amino acids and sugars from the endosperm to the embryo for rapid seed germination (Hussain et al., 2017) and thus improves seedling emergence and crop establishment upon sowing under field conditions (Singh et al., 2015; Mishra et al., 2017).
       
However, the effect of hydropriming varies with crop (Sarvjeet et al., 2017 in rice, finger millet, chickpea), varieties within a crop (Carver et al., 2014; Nakao et al., 2020), seed density (Nakao et al., 2020), drought conditions (Carver et al., 2014) and period of hydropriming (Carver et al., 2014; Mishra et al., 2017). For effective hydropriming, the duration of the priming is crucial, but in general, overnight soaking is followed. Such a longer priming duration might lead to seed deterioration and a shorter priming duration will be ineffective in initiating the germination process. Although genotypic differences exist for the priming effect (Nakao et al., 2020), as a proof of concept, considering one popular variety for each one of the major crops could be meticulous. Therefore, the objective of the present investigation was to determine the critical and optimal priming duration in the predominant pulse (pigeon pea), oilseed (sunflower) and cereal (>45% area is upland rice) crops for better seedling growth and seedling vigour index.

The experiments were conducted in the laboratory (temperature of 26°C and 65% RH) and the field facility of the Department of Crop Physiology, University of Agricultural Sciences, GKVK, Bengaluru (May 2020 to August 2020). The fresh seeds of pigeon pea (cv. BRG-5), sunflower hybrid (KBSH-78) and rice (MTU-1001) were hydro-primed (10g in 100 ml water in a 200 ml beaker) in five replications in a completely randomized design. At every hour interval of priming, seeds were strained out of the beaker using a mesh strainer, gently wiped with tissue paper to remove the adhered water, took the fresh weight and placed back into the beaker. The rate of imbibition and the cumulative imbibition were calculated. In another set, seeds were hydro-primed for 0 to 12 h at hourly and at 24 h, the primed seeds were shade-dried to their original dry weight. Such seeds were sown in the field in three replications with protective irrigation during July 2020 for 15 days. At 15 days after sowing, the seedling length and weight were recorded to calculate the seedling vigour index. ANOVA was performed using OPSTAT statistical software (Sheoran et al., 1998).
 

Seed germination and seedling emergence are the prominent stages of crop establishment, plant population and productivity (Nakao et al., 2018). In the climate change scenario, frequent drought is one of the major threats to seed germination and crop establishment that depends on rainfall for seed germination in arid and semi-arid regions (Angadi and Entz, 2002). The predominant crops like pigeon pea, sunflower and upland rice are drought-prone under rainfed conditions. One of the approaches to mitigate the drought stress during the initial stage of the crop could be by adopting the high vigour seeds or improving the seedling vigour (Matsushima and Sakagami, 2013). Among these two, hydropriming or seed hardening is preferable to enhance the seedling vigour and drought tolerance during the seedling stage (Rajashekara et al., 1970). The advantage of seed priming is that the hydropriming can also be extended for low vigour seeds. The increase in seedling vigour due to priming depends on the rapid imbibition of the water by the seed for subsequent physiological processes to improve the seed germination (Singh et al., 2015). Imbibition increases the hydrolytic enzymes to break the complex molecules like sugars, proteins and fats into simpler sugars like glucose for increased respiration rate and seed germination (Woodstock, 1988).
 
Effect of hydropriming duration on imbibition percentage
 
The rate of imbibition in the first hour of water soaking was very high (130%) in pigeon pea and relatively slower in sunflower (40%) and rice (40%; Fig 1; Maqueo et al., 2020). Such higher imbibition rates could be due to the exceptionally lower water potential (matric potential) in the dry seeds at the initial stages, activation of protein synthesis and mitochondrial repairs (Vari and Dadlani, 2010). Also, the high imbibition rate in pigeon pea is due to the thin seed coat compared to sunflower and rice (Fig1; Woodstock, 1988). The imbibition rate was decreased to nearly 10% in sunflower and rice within 3 to 4 hours, whereas it was 5 hours in pigeon pea. The extended hour of imbibition in pigeon pea is due to higher protein and absorbs more water with higher swelling. The imbibition rate reached zero (saturation) by 11 hours (pigeon pea) and 12 hours (sunflower and rice). Once again, in the third phase of the germination process, a marginal increase in imbibition rate from 12 to 24h was observed in pigeon pea and sunflower, whereas it was very high (35%) in the case of rice. The increase in imbibition could create high turgor pressure to enable the rupture of the seed coat and protrusion of radical (Vari and Dadlani, 2010). These results demonstrate that the rate of water uptake will be high during the early hours of seed imbibition and remains similar in the rest of the phases in sunflower and pigeon pea. However, rice followed a rapid increase in imbibition rate during the third phase of germination for radical protrusion. 
 

       
However, the seed germination depends on the cumulative critical imbibition percentage of 30-40%. It attained within 2-3 hours of priming in pigeon pea and sunflower (Table 1; Priya Reddy and Deshpande, 2020) and 24h in rice to reach 25% imbibition (Fig 1). As pigeon pea and sunflower non-endospermic seeds with cotyledons will have high protein content, thus rapid imbibition compared endospermic seed the rice (Zhao et al., 2018).  The cumulative imbibition was increased gradually up to 24h priming and reached 120% (pigeon pea), 90% (sunflower) and 25% (rice) (Fig 1). Similar cumulative imbibition of 81.5% by 24h has been reported in sunflower (Priya Reddy and Deshpande, 2020). The higher cumulative imbibition in pigeon pea and sunflower could be due to higher protein content when compared to carbohydrate-rich rice (Zhao et al., 2018). Although the required critical imbibition (40%) reached in 2-3h in sunflower and pigeon pea, the long period of soaking period up to 7-8 hours could be apt, as the seeds will be shade dried back original seed moisture content, such priming will induce immediate absorption of water under field conditions. These results imply that the maximum priming period can be 7-8 hours and seeds (pigeon pea) with thinner seed coats and higher protein content will accumulate more water in the seed upon priming. In sunflower, it could be due to higher protein content and a large air space between the hull and kernel. The rice being a carbohydrate-rich seed with less protein, the maximum imbibition was less (25%).
 
  
 
Effect of hydropriming duration on seedling growth and vigour
 
In pigeon pea, 2-3h priming increased the seedling length, with the highest in the 8-hour priming (25.7 cm; Table 1; Fig 2). Similarly, 1 to 2 hours of hydropriming shown to increase the seed germination, seedling length and seedling vigour index (Sajjan et al., 2017; Ashok kumar et al., 2017) and also by 4 hours priming, however over-priming above 4h affected these seedling parameters (Carver et al., 2014). A 6h hydropriming has been shown to increase the seed germination, seedling length, seedling vigour, chlorophyll content and enzymes like nitrate reductase, nitrite reductase, catalase, peroxidase, superoxide dismutase leading to increased seedling vigour (Tiwari and Agarwal, 2021). In contrast, hydropriming of pigeon pea seeds for 8-12 hours was reported to increase the field emergence, with an increased nodule number and nodule weight at 40 to 90 days after sowing (Mishra et al., 2017). The present study confirms that the maximum seedling length and SVI could be attained by 7-8h of priming, beyond which it declines. The seedling fresh weight (931 mg/seedling), dry weight (165.5 mg/seedling) and SVI (4068) were also higher in 7h priming. The SVI was reduced by 8.2% and 5.7%, respectively in 12 and 24h of priming compared to 8h priming (Table 1). Therefore, the critical imbibition period could be 2-3 h and to a maximum of 7-8 hours could be appropriate for pigeon pea, beyond which the thin seed coat will be ruptured, causing damage to the embryo and endosperm. 
 
 
       
In sunflower, hydropriming the seed for 8h significantly increased the seedling length (13.08 cm) compared to no-priming (11.95 cm) and significantly reduced by 24h of priming (11.38 cm, Table 2; Fig 2). The SVI was highest (3322) in 8h priming and reduced significantly to 2339 by 24h priming duration (Table 2). Similarly, priming for 6h positively influenced the seed germination and seedling length (Lekic et al., 2015). The increase in germination and SVI with priming was ascribed to rapid uptake of water, higher dehydrogenase activity, lower leakage of solutes (Shanthala et al., 2013); increased catalase, peroxidase, glutathione reductase, proline content, with a reduced MDA (Ocvirk et al., 2020); and increased reducing sugars required for respiration (Bourioug et al., 2020). An 8h hydropriming was found to reduce the blooming duration with enhanced chlorophyll, carotenoids, stomatal conductance, transpiration, photosynthetic rate and soluble sugars and yield (Bourioug et al., 2020). Hydropriming for 12 h (Catiempo et al., 2021; Pavithramata et al., 2023) and 18h (Shanthala et al., 2013; Jovicic et al., 2022) was also found to increase the seedling length, dry weight and vigor index in sunflower. In contrast, no improvement in seedling parameters was observed by priming for 18 h (Catiempo et al., 2021), 12 h (Farahani et al., 2011) and 16h (Bourioug et al., 2020), rather yield was decreased by 16.7% with 16h priming (Bourioug et al., 2020). Therefore, priming for beyond 8h appears to be negative (Table 2) and 6-8h hydropriming duration could be appropriate for maximum SVI in sunflower. The critical imbibition period could be 2 hour and a maximum can be 8 hours for sunflower.
 

       
In rice, hydropriming did not increase the total seedling length significantly, but the root length was increased significantly in 6h priming compared to the no-priming (Table 3). The SVI was significantly higher with 3h of imbibition compared to the control, beyond which there was no significant improvement (Table 3). In this direction, 6h hydropriming was reported to enhance germination and vigour index, especially in drought-tolerant varieties (Ranmeechai et al., 2022) and even 24h soaking (Ramesh and Singh, 2006). In the present study, priming for 12 or 24 h significantly reduced the fresh weight, dry weight of seedlings and the SVI evidenced by phenotypic expression (Fig 2). The prolonged priming was reported to lead to seed deterioration (Ren et al., 2023) which could be due to anaerobic conditions during imbibition thus reduced respiration and hence more than 7-8h hydropriming had no advantage. Furthermore, during re-drying of primed seed, the higher respiration might have used the available carbohydrates available and hence decreased growth in 24 soaking periods.
 

       
The shoot-to-root ratio was lesser in pigeon pea and sunflower compared to the rice (Table 1, 2, 3), suggesting the higher effect of hydropriming on root growth in pigeon pea and sunflower, the rainfed crops, where root growth is crucial for seedling survival in contrast to rice. The phenomenon is evidenced by a higher standardized range for root compared to the shoot in all three crops with the phenotypic expression (Fig 2). The priming could give a head start due to the stimulatory effect conserved during re-drying (Catiempo et al., 2021), that induces the de novo synthesis of hydrolases, breaking down food reserves and efficiently translocation to the growing embryo (Hussain et al., 2017). Another hypothesis could be memory due to priming, for rapid imbibition of water upon sowing in the field (Chen and Arora, 2013). Although the imbibition period expected to vary with the genotype, considering the popular varieties, as a proof of concept, the critical imbibition period could be 3h and can be extended to 7-8h for maximizing the seedling vigour in pigeon pea, sunflower and rice.

Seedling establishment is a major impediment in rainfed crops like pigeon pea, sunflower and upland rice. Critical imbibition of 30-40% required for optimum seed germination will be attained within 3-4 hours of hydropriming, however, the seedling establishment could be better by 7-8 hours of hydropriming in pigeon pea, sunflower and rice. Therefore, hydropriming is a simple, inexpensive and easy technique to enhance the crop stand.

No conflict of interests regarding the publication.