Effect of Nutripriming with Boric Acid, Zinc Sulphate Heptahydrate and its Combination on Germination Physiology of Lentil (Lens culinaris L.) cv. HUL 57

Lentil (Lens culinaris) belongs to the Leguminaceae (Fabaceae) family, nutritious amongst the rabi pulses, used as a cheap source of protein and cultivated in rainfed areas. Due to changing climatic conditions plants are suffering from various environmental stresses. Seed priming is an alternating way out which have the ability to activate the pre-germination metabolism without radicle protrusion and improves the germination rate, seedling establishment and overall growth and productivity of crop plants (Mondal and Bose, 2019). Moreover, Ghassemi-Golezani et al., (2013) noted that hydro-priming of lentil seeds increased the height of the plant, pods and seeds number per plant, biological and grain yield and harvest index in comparison to the non-primed one. For instance, Toklu (2015) observed that ZnSO₄, GA₃ and PEG-6000 priming treatments having a positive effects of germination rate, germination percentage, yield component and grain yield of lentil plants. However, Kumar et al., (2019) reported that seed priming having the potential to increase the growth, yield and seed quality characteristics of lentil which enhance the income of farming communities. In addition, Bhateshwar et al., (2020) noted that the maximum germination percentage, number of branches per plant, seeds per pod, 1000 seeds weight, yield per plant and yield per ha were increased when seeds were primed with neem leaf extracts. Scientific reports related to seed priming depicted that it is an important contributory factor for the improvement of synchrony and speed of germination and also establish the seedlings for better growth near future  Ghassemi-Golezani et al.(2008), Ghassemi-Golezani et al.(2013), Bhateshwar et al. (2020)  in lentil.    
       
Based on this documentation, the present study aimed to investigate the effects of different micronutrients like boric acid, zinc sulphate and its combination in a various concentrations range for the purpose of seed priming, during the time of seed germination, emergence, seedling establishment and growth of lentil (cv. HUL 57). 
 

The whole experiment was carried out in the Seed Physiology Laboratory, Department of Crop Physiology, Visva-Bharati in the year 2014-15 and 2015-16. Healthy and bold seeds were surface sterilized with 0.01% HgCl₂ solution. For priming, the sterilized lentil seeds were soaked in different concentrations/ combinations of boric acid (H₃BO₃) and zinc sulphate heptahydrate (ZnSO₄.7H₂O) solution (Treatment details were as follows: (Unit: in mM) [Boric acid: B₀ = Control, B₁ = 2, B₂ = 4, B₃ = 6, B₄ = 8, B₅ = 10, B₆ = 15, B₇ = 20, B₈ = 30]; [Zinc sulphate heptahydrate: Zn₀ = Control, Zn₁ = 2, Zn₂ = 4, Zn₃ = 6, Zn₄ = 8, Zn₅ = 10, Zn₆ = 15, Zn₇ = 20, Zn₈ = 30]; [Combination: T₀ = Control; T₁= 2 mM ZnSO₄.7H₂O + 4mM H₃BO₃; T₂ = 2mM ZnSO₄.7H₂O + 6 mM H₃BO₃; T₃= 4 mM ZnSO₄.7H₂O + 4 mM H₃BO₃ and T₄= 4 mM ZnSO₄.7H₂O + 6 mM H₃BO₃]) for 12 h. The seeds without any treatment referred as control (non-primed). Then 50 dried seeds of each treatment along with the control one were taken and placed in different petridishes. The petridishes of 3.0-inch diameter were used to conduct germination physiology of lentil under normal light and room temperature (25±2°C) conditions. The parameters considered for the experiment was germination percentage (ranging from 18 to 96h), seedling growth in terms of radicle and plumule lengths, fresh and dry weights of radicle and plumule at different time intervals (ranging from 48 to 96 h) during both the consecutive years i.e., 2014-15 and 2015-16 (Mondal et al., 2011, Kumar et al., 2016). The experiment was arranged in 3 replications and the data were analyzed statistically by following completely randomized design.
 
 

Table 1 depicted the germination percentages of boric acid primed and non-primed lentil seeds at different studied hours (18, 24, 36, 48, 72 and 96 hours) during both 2014-15 and 2015-16. The results showed that the germination percentage was increased with increasing duration and varied significantly among the different priming treatments. The highest germination percentage was recorded after 18 h of sowing in treatment B₂ i.e., 4 mM H₃BO₃ (91% and 89%). The germination percentage was increased gradually upto 48h and  thereafter it did not change much.


       
Table 2 denoted the germination percentages of lentil seeds primed with Zinc sulphate heptahydrate at different intervals of time during both 2014-15 and 2015-16 Seed priming with 2mM ZnSO₄, 7H₂O (Zn₁) recorded the highest germination percentage (89.0-100%) in all the studied hours during both the years; followed by Zn₂ (83.0 -99.7%) and Zn₃ (87.0-97.0%) and showed statistically at par results.


       
In Table 3, the highest germination percentage (88.1-99.7%) was obtained by T₁ (2mM ZnSO₄.7H₂0 + 4mM H₃BO₃) in all the studied hours followed by other treatment combinations and recorded significantly greater germination percentage than that of T₀ (87.3-94%) and T₄ (86.3-95.3%) in both years. The results showed a decreasing tendency of germination percentage due to increasing the concentration of priming treatments.


       
Fig 1 revealed the fresh weight of plumule and radicle obtained from H₃BO₃ primed and non-primed seeds at 48, 72 and 96h after sowing. Treatment B₂ recorded the highest fresh weights of plumule (4.4, 7.2 and 10.9 mg in 2014-15 and 8.5, 11.1 and 13.1 mg in 2015-16 at 48, 72 and 96 h respectively). Whereas, fresh weight of radicle showed similar trends to that of fresh weight of its plumule.


       
Fig 2 noted the data regarding ZnSO₄.7H₂O primed and non-primed plumules and radicles fresh weight, where Zn₁ showed the highest value of fresh weights of plumule (5.0, 11.8 and 13.8 mg in 2014-15 and 5.6, 9.4 and 12.1 mg in 2015-16 at 48, 72 and 96 h respectively) and radicle (11.4, 15.3 and 17.5 mg in 2014-15 and 13.8, 15.3 and 16.8 mg in 2015-16 respectively) which was significantly greater than that of all other priming treatments.


       
The treatment T₁ showed the highest value of fresh weight of plumule (7.18, 13.33 and 17.75 mg in 2014-15 and 10.17, 14.55 and 17.74 mg in 2015-16 at 48, 72 and 96 h, respectively) and radicle (13.75, 17.75 and 20.50 mg in 2014-15 and 14.30, 17.93 and 19.72 mg in 2015-16 at 48, 72 and 96 h, respectively), followed by T₂ at all the stages. The control (T₀) treatment recorded the lowest value of fresh weight of plumule and radicle (Fig 3).


       
In Fig 4, the highest dry weights of plumule (1.90, 3.91 and 4.30 mg in 2014-15 and 1.35, 3.41 and 4.24 mg in 2015-16 at 48, 72 and 96 h respectively) and radicles (3.77, 5.72 and 7.02 mg in 2014-15 and 2.86, 2.27 and 6.40 mg in 2015-16 at 48, 72 and 96 h respectively) were recorded in B₂ followed by other treatments.


       
Fig 5 recorded that the seeds primed with Zn₁ (2mM ZnSO₄. 7H₂O) recorded the maximum plumule (1.15, 2.42 and 3.02 mg in 2014-15 and 0.97, 2.15 and 2.74 mg in 2015-16 at 48, 72 and 96 h respectively) and radicle dry weights (i.e., 2.99, 3.52 and 4.23 mg in 2014-15 and 2.85, 3.39 and 3.94 mg in 2015-16 at 48, 72 and 96 h respectively) which was significantly greater than treatments at all the stages during both the years. The treatment Zn₈ was recorded the lowest value of dry weight. Dry weight of plumule and radicle showed similar pattern just like fresh weight obtained from 2 mM ZnSO₄.7 H₂O + 4 mM H₃BO₃ at 48, 72 and 96 h, which showed statistically significant results among the other treatments presented in Fig 6.




       
In Fig 7, the graphs showed that the length of plumule decreased gradually with the increasing concentrations of H₃BO₃. It was observed that treatment B₂ showed the highest plumule length (6.20, 7.46 and 8.57 mm in 2014-15 and 5.41, 6.48 and 7.3 mm in 2015-16 at 48, 72 and 96 h respectively) and radicle length (9.42, 11.52 and 12.62 mm in 2014-15 and 9.42, 11.60 and 12.63 mm in 2015-16 at 48, 72 and 96 h respectively) which was significantly greater than that of all other nutripriming treatments.


       
Fig 8 showed the plumule and radicle length and the result showed that Zn₁ (2 mM ZnSO₄. 7H₂O) obtained highest plumule length (4.6, 6.0 and 7.6 mm in 2014-15 and 5.0, 6.5 and 7.7 mm in 2015-16 at 48, 72 and 96 h) and radicle length (11.72, 16.93 and 19.63 mm in 2014-15 and 12.90, 18.68 and 21.60 mm in 2015-16 at 48, 72 and 96 h respectively) it was significantly greater than that of all other priming treatments in all the studied hours.  


       
In Fig 9 treatment T₁ recorded the highest plumule (5.17, 6.25 and 7.08 mm in 2014-15 and 5.50, 6.72 and 7.41 mm in 2015-16 at 48, 72 and 96 h, respectively) and radicle lengths (12.48, 15.25 and 17.96 mg in 2014-15 and 13.68, 16.22 and 18.70 mg in 2015-16 at 48, 72 and 96 h, respectively) and which were statistically significant than that of treatment T₀ and T₄.


       
Erratic rainfall patterns and temperature extremes create a stressful situation specially for germinating seeds and young seedlings as a result there is slow and non-uniform germination with a compromised vigour was observed in lentil (Lens culinaris Medik.) and finally yield potential is also affected (Ghasemi-Golezani et al., 2013).
       
In present investigation with lentil Var. HUL 57, while primed with alone/ different combinations of zinc sulphate heptahydrate and boric acid were found to improve the germination percentage as compared to control. Whereas, the lower dose of zinc sulphate heptahydrate and boric acid were found effective for priming treatment of lentil seeds in comparison to higher doses. In this context, the lower doses of ZnSO₄. 7H₂O and H₃BO₃ were taken into consideration and apply it in combination as priming agent to get the best possible result in terms of early growth phase of lentil. The variety HUL 57 showed improved germination percentage, better seedling establishment and seedling vigour while primed with these micronutrients. Likely, an increase in germination percentage/ velocity/ rate in micronutrient primed seeds were recorded by various scientists’ time to time with a number of field crops, vegetable and ornamental plants as reported by Mondal and Bose (2019). The same has been observed in the present investigation where in respect to control all primed sets enhanced the plumule and radicle lengths, fresh and dry weights of growing seedlings. The priming technology offered the synchronized germination and improved seedling vigour which has been noticed by a number of workers (Farooq et al., 2006; Yuan-Yuan et al., 2010; Ella et al., 2011; Mondal et al., 2011). The critical scrutinization of the results for all the above-mentioned parameters showed that the application of zinc sulphate heptahydrate and boric acid both have an optimum concentration where the maximum attainments of the values were observed in present case. Similarly, Mondal and Bose (2022) reported that in plant system has the capacity to act as per the concentration of the chemicals, which are beneficial for their growth, otherwise the level more than that may be either toxic or inhibitory. In another study, Farooq et al. (2006) noted that the optimum concentration of zinc sulphate heptahydrate and boric acid either used separately or in combined form during the time of seed priming may improve the homeostasis by maintaining the suitable criteria for the germinating seeds and seedling. In addition, Ghassemi-Golezani et al., (2008) revealed that a useful and simplified technique i.e., hydropriming is responsible for enhancing the seedling emergence rate and percentage of lentil. Whereas, (Farooq et al., 2019) observed that under normal and water deficit condition, osmopriming improved the lentil performance by improving early and synchronized emergence, better accumulation of sugar and Calcium which have the potential to reduce the oxidative damage and resulting better seedling growth and biomass production. In context to this, Toklu et al., (2015) provide conclusive evidence that in terms of germination properties, various plant characteristics, components of grain yield and grain yield in lentil; seed priming with GA₃, PEG and ZnSO₄ should be considered as farmers recommendation and further research is needed in this respect.
       
Moreover, different experiment was done by various scientist in support of lentil seed priming; where Singh et al., (2017) indicates a possibility of enhancing productivity in lentil by using PGR (Cycocel, GA₃ and IAA) as priming agent for the improvement of parameters related to growth and yield. Whereas, Bhateshwar et al., (2020) reported that by using botanical extracts for seed priming like neem leaf extract, castor oil, ginger extract and onion were responsible for the betterment of the growth and yield in lentil.
 
 

Seed germination and seedling establishment of lentil can be inhibited by multifarious environmental conditions and the responses are varied according to the species and cultivars. In a nutshell we can conclude that seed priming with 4mM boric acid, 2 mM zinc sulphate heptahydrate and zinc sulphate heptahydrate (2 mM) + boric acid (4 mM) as single or in combination may be used to improve the germination physiology as well as seedling growth of lentil, in the field conditions during erratic climatic conditions. However, in respect to environment and farmers seed priming with micronutrients is a safe, eco-friendly, cost effective and sustainable approach.