Seed Hardening in Relation to Seedling Quality Characters of Green Gram (Vigna radiata L.)

Pulses play an important role in maintaining the soil fertility in sustainable manner in addition to being an important source of human food and animal feed. Mung bean is one of the important pulse crops and rank third in area and production after pigeon pea and chickpea. Mung bean (Vigna radiata L.) belongs to the family Leguminoceae (Fabaceae). It is grown in almost all parts of the country over a wide range of agro-climatic conditions. India is the largest producer of mung bean in the world. It accounts for almost 65 per cent of area and 54 per cent of production of the world. Green gram is a drought resistant crop and suitable for dry land farming and predominantly used as an intercrop with other crops. It is a very good catch crop in summer and can be grown very well in this season. Mung bean is a short duration, low input requiring crop that matures in 65 to 80 days, photo and thermo-insensitive in nature. It adds about 40 kg N ha^-1 in the soil by fixing the atmospheric N which is subsequently beneficial to succeeding crops. Mung bean is typically a quantitative short day plant. The productivity of mung bean is low.
       
Efforts made to maximize yield, is largely hampered by adverse effect of abiotic stresses such as salinity and drought. These effects cause a huge loss due to low yield and failure of the crop to establish in some cases. Pre-sowing hardening seed treatment is an easy, low cost and low risk technique and also an alternative approach recently used to overcome the effect of abiotic stresses in agricultural production. It is found to be efficient in improving seed emergence and growth of crops. Sankar Ganesh et al., (2013), Bewley and Black (1982) and JunMin et al., (2000) reported clearly that the hardening treatment enhance seeds vigour by protecting structure of the plasma membrane against injury during stress. It is a well established fact that, pre-soaking seeds with optimal concentration of phytohormones enhance their germination, growth and yield of some crop species under condition of environmental stress by increasing nutrient reserves through increased physiological activities and root proliferation (Bozeuk, 1981).
       
Rapid and uniform field emergence is the essential pre-requisites for increasing the yield and improving quality. To overcome the moisture problem prevailing in dry land agriculture, Henckel (1964) recommended the seed hardening techniques to alleviate the moisture stress condition. According to Prajapati et al., (2017) hardening induces early germination, better root and seedling growth, reduces seedling mortality, increases crop population and thereby enhancing the yield potential of the crop varieties. Hardening of seeds resulted in the absorption of more water due to increase in the elasticity of cell wall and development of a stronger and efficient root system (Krishnasamy and Srimathi, 2001). Increased germination rate and uniformity have been attributed to metabolic repair processes occurring during imbibitions, by enhancing metabolites (Basra et al., 2005) and reduced imbibitions lag time (Bradford, 1986), quick recovery of hardened plants from wilting than those from untreated plants, induction of resistance to salinity and drought situation, ability of seeds to withstand higher temperature for prolonged period, slight acceleration of flowering and capacity to compete more efficiently with weeds due to early emergence and finally resulting in higher yield.
       
Considering the constraints in the production potential of mung bean it is worthwhile to study the influence of different seed hardening treatments on the production potential of mung bean. It is also of utmost importance to understand the physiological basis of yield variation due to seed hardening of various growth regulators and chemicals.

The present work was carried out at the Department of Plant Physiology, Anand Agricultural University, Anand to study the effect of seed hardening treatments on morpho-physiological and seed quality characters in green gram during summer season of 2015-16 and 2016-17. The laboratory trial was laid out in CRD with three repetitions. Seeds of mung bean var. GAM- 5 were imposed with the following seed treatments.
       
T₁: CaCl₂ @ 2%, T₂: 500 ppm Cycocel, T₃: 1000 ppm Cycocel, T₄: 25 ppm NAA, T₅: 50 ppm NAA, T₆: Water soaked control and T₇: Absolute control.
       
A day before sowing, seeds of mung bean variety GAM-5 were soaked for three hours separately in solutions of CaCl₂ @ 2%, Cycocel 500 ppm, Cycocel 1000 ppm, NAA 25 ppm, NAA 50 ppm and distilled water. Later seeds were dried under shade for overnight and sown on moist germination paper next day. In each treatment 100 seeds were sown and this was repeated three times. The rolled germination papers were kept in seed germinator at 25°C at proper moisture. The seeds were inspected at interval and moistened regularly with water.
       
Ten seedlings from each treatment were selected randomly for the purpose of recording morpho- physiological and seedling quality characters. The germination was calculated on the basis of normal seedlings counted and expressed in percentage. Seeds which remain hard at the end of prescribed test because they have not absorbed water due to an impermeable seed coat. The hard seeds were counted on the final day and expressed in percentage.
       
Root and shoot length were recorded by measuring the length using a meter scale and the mean value was expressed in centimeter. To record seedling fresh weight ten seedling were counted randomly from the germination test, cut free from their cotyledon and weighted while still moist. Their weights were recorded in gram. The seedlings used for recording fresh seedling weight were kept to float on distilled water in a petri dish and allowed to absorb water for four hours. After four hours, the seedlings were taken out and their surface was blotted gently to remove excess water. The weight of the turgid seedlings was recorded and expressed in gram. These randomly selected ten seedling samples were separated into root and shoot and dried in oven at 85°C for 24 hours. After drying the samples were used to dry weight measurement.
       
Seed Vigour Index I in terms of length was determined by multiplication of germination percentage with seedling length. The vigour indices were calculated using the following procedure suggested by Abdul-Baki and Anderson (1973) and expressed in whole number.
   
Seed Vigour Index II in terms of mass was determined by multiplication of germination percentage with seedling dry weight suggested by Abdul-Baki and Anderson (1973).
   
The relative water content was estimated by the method of Barrs and Weatherly (1962). The relative water content (RWC) of seedling was calculated by the following formula and expressed in percentage.
   
The moisture content per seedling is calculated by the following formula and expressed in percentage.
 
The root shoot ratio on dry weight basis per seedling was calculated by the following formula
 
The root shoot ratio on length basis per seedling was calculated by the following formula
   
The data were statistically analyzed using ANOVA.

The result of different seed hardening treatments revealed that all physiological characters were significant in different seed hardening treatments, indicating positive influence on these characters. The data on seed germination and hard seed per cent is presented in Table 1 and 2 respectively. Among the various treatments, pre-sowing seed hardening with CaCl₂ at 2% (T₁) recorded significantly the highest seed germination and lowest hard seeds (91.00% and 7.17%, respectively) as compared to control (78.17% and 18.33%, respectively) during both season’s pooled analysis. However, it remained at par with the treatments Cycocel 1000 mg/L (T₃) (89.33% and 8.33%, respectively). The probable reason for early and higher germination of soaked seeds may be due to completion of pre-germinative metabolic activities making the seed ready for radical protrusion in mung bean reported by Manjunatha and Dhanoji (2011) and Sujatha (2014), in chickpea. This might be due to a number of physio-chemical changes occurred in seed that modify the protoplasmic characters and thereby increased physiological activity of embryo and associated structures development for efficient seedling germination. These results are in conformity with the finding of Srimathi and Sujatha (2006), Surulirajan (2007), Patil et al., (2014) in cotton, Prajapati et al., (2017) in black gram, Patel et al., (2017) and Bhadane et al., (2019) in green gram.




       
The data on fresh, turgor and dry weight of seedling is presented in Table 1. On pooled basis, the treatment 2% CaCl₂ (T₁) recorded significantly the highest seedling fresh weight (4.567 g) as compared to rest of the treatments. Increase in seedling fresh weight was due to increased shoot fresh and root fresh weight combinely. This might be due to increase in the rate of absorption of water and available nutrients. Similar findings are also reported by Mandal and Basu (1987) in wheat, Patil (1987) in sorghum, Kinjal (2017) in urd bean and Patel et al., (2017) in mung bean.
       
Among the treatments, 2% CaCl₂ (T₁) recorded significantly highest seedling turgor weight (4.793 g, 4.918 g and 4.855 g) than both the control treatments. However, the treatment T₁ remained at par with NAA 50 mg/L (T₅) (4.699, 4.755 and 4.727 g) during the year 2015-16, 2016-17 and on pooled basis, respectively. Similarly, the treatment of Cycocel 1000 mg/L (T₃) was also found at par (4.638 and 4.709 g) with T₁ during the year 2015-16 and 2016-17, respectively but not on pooled basis. Significantly the lowest seedling turgor weight (3.456, 3.574 and 3.515 g) recorded during both the years and on pooled basis, respectively in absolute control (T₇).
       
The treatment 2% CaCl₂ (T₁) recorded the highest seedling dry weight (0.292 g); whereas, the minimum in absolute control (T₇) (0.199 g) in both years pooled. The increased shoot and root dry weight may be due to enhanced lipid utilization through glyoxalate cycle. The increase in shoot and root dry weight increased seedling dry matter. These results are in accordance with the findings of Patil (1987) in sorghum, Corleto et al., (1977) in green gram, Mandal and Basu (1987) in wheat, Punithavathi and Palaniswamy (2001) in finger millet, Prakash et al., (2013) in rice, Kinjal (2017) in black gram and Patel et al., (2017) in green gram.
       
The root, shoot dry weight and relative water content is reported in Table 2. The treatment NAA 50 mg/L (T₅) recorded significantly highest root dry weight (0.071, 0.072 and 0.071 g) as compared to rest of the treatments during both the years and on pooled basis, respectively. Significantly the lowest root dry weight (0.038, 0.038 and 0.038 g) was recorded in absolute control (T₇). The increased root dry weight observed may be due to more number of roots, enhanced lipid utilization through glyoxalate cycle and enabling to produce relatively more quantity of dry matter. The seed hardening with 2% CaCl₂ (T₁) recorded significantly highest shoot dry weight (0.242, 0.251 and 0.246 g) as compared to rest of the treatments and remained at par with Cycocel 1000 mg/L (T₃) (0.230, 0.230 and 0.230 g) during the years 2015-16, 2016-17 and in pooled analysis, respectively as well as with the Cycocel 500 mg/L (T₂) treatment during the year 2016. Significantly lowest shoot dry weight (0.155, 0.168 and 0.161 g) were recorded in absolute control (T₇). These results are in accordance with the findings of Patil (1987) in sorghum, Corleto et al., (1977) in green gram, Mishra and Dwivedi (1980) in wheat, Mandal and Basu (1987) in wheat, Punithavathi and Palaniswamy (2001) in finger millet, Prakash et al., (2013) in rice seed, Kinjal (2017) in black gram and Patel et al., (2017) in green gram.
       
The treatment NAA 25 mg/L (T₄) showed the highest (93.94%) relative water content followed by treatment 2% CaCl₂ (T₁) (93.69%), Cycocel 1000 mg/L (T₃) (93.23%), while minimum in absolute control (T₇) (91.08%) in pooled analysis. Relative water content (RWC) is a measure of the amount of water present in the leaf tissue in relation to turgid condition. These results are in accordance with Patil (1987) who revealed that pre-sowing seed treatment with 2% CaCl₂ recorded higher RWC in sorghum. Similar results were also reported in wheat by Amaregouda et al., (1994)  and Manjunatha (2007) in chick pea.
       
The data on root, shoot, seedling length and moisture content is presented in Table 3. Significantly longer root length (14.55, 14.73 and 14.64 cm) were recorded in seed hardening with CaCl₂ at 2% (T₁) as compared to control and remained at par with the treatment Cycocel 1000 mg/L (T₃) (13.62, 13.52 and 13.57 cm) during both the individual years as well as in pooled basis, respectively. Whereas, significantly the shortest root length (7.78, 7.93 and 7.86 cm) was recorded in control. This might be due to a number of physio-chemical changes within the cytoplasm including greater hydration of colloids, higher viscosity and elasticity of the protoplasm, increase in hydrophilic colloids and decrease in lipophilic colloids, increase in the temperature required for protein coagulation and increase in bound water content. Root plays a major role as far as moisture extraction and nutrient absorption are concerned. Seed hardening with 2% CaCl₂ was found to increase root growth even at the seedling stage. The results are in concurrence with the earlier findings of De et al., (1982), noticed in wheat, Patil (1987)  in sorghum, Corleto et al., (1977) in green gram, Punithavathi and Palaniswamy (2001) in finger millet, Kinjal (2017) in black gram and Patel et al., (2017) in mung bean.
 

       
Seed hardening with CaCl₂ at 2% (T₁) recorded significantly higher shoot length (10.43, 10.51 and 10.47 cm) as compared to control treatment. It was remained at par with the treatment Cycocel 1000 mg/L (T₃) (9.82, 9.74 and 9.78 cm) during the years 2015-16 and 2016-17 as well as in pooled analysis, respectively. Significantly the shortest shoot length (6.84, 6.97 and 6.91 cm) was noticed in absolute control (T₇). This might be due to increase in the rate of absorption of water and available nutrients and thereby resulting in better growth. The results obtained from this study are in accordance with those reported by Patil (1987), Corleto et al., (1977), Mandal and Basu (1987) and Punithavathi and Palaniswamy (2001), Kinjal (2017), Patel et al., (2017), Bhadane et al., (2019) in sorghum, green gram, wheat, finger millet, black gram and green gram, respectively.
       
Significantly the highest seedling length was observed under the treatment 2% CaCl₂ (T₁) (24.98 and 25.23 cm) and statistically at par with Cycocel 1000 mg/L (T₃) (23.43 and 23.26 cm) during the year 2015-16 and 2016-17, respectively. In pooled analysis, the treatment 2% CaCl₂ (T₁) (25.11 cm) recorded significantly highest seedling length than rest of the treatments. While minimum seedling length was observed in untreated absolute control (T₇) (14.62, 14.90 and 14.76 cm). The increase in seedling length results from increased shoot and root length. This might be due to increase in the rate of absorption of water and available nutrients and thereby resulting in better growth of seedlings. The results obtained from this study are in accordance with those reported by Patil (1987), Corleto et al., (1977) and Patel et al., (2017), Mandal and Basu (1987) and Punithavathi and Palaniswamy (2001), Kinjal (2017) in sorghum, green gram, wheat, finger millet and black gram, respectively. The treatment NAA 25 mg/L (T₄) recorded highest moisture content (93.89%) on pooled basis, but results were non significant. Similarly, minimum moisture content (93.53%) observed in the treatment Cycocel 500 mg/L (T₂) on pooled basis.
       
The data regarding seedling vigour indices and root shoot ratio is shown in Table 4. All the seed hardening treatments significantly increased seedling vigour index I than non hardened absolute control treatment (T₇). The treatment 2% CaCl₂ (T₁) recorded significantly highest seedling vigour index I (2229, 2336 and 2282) than rest of the treatments. Significantly lowest seedling vigour index I (1111, 1197 and 1154) was observed during the year 2015-16, 2016-17 and on pooled basis, respectively in absolute control (T₇). Increase in seedling vigour index I on length basis by seed hardening treatments was due to higher seed germination and increased shoot and root length. The treatment 2% CaCl₂ (T₁) recorded significantly the highest seedling vigour index II on mass basis (2.657); whereas, the minimum was in treatment of absolute control (T₇) (1.558). The increase in seedling vigour index II on mass basis with seed hardening treatments was due to higher seed germination per cent as well as increase in shoot and root dry weight. The results obtained from this study were in accordance with those reported by Rangaswamy et al., (1993) in red gram, Punithavathi and Palaniswamy (2001) in finger millet, Surulirajan (2007) and Kinjal (2017) in black gram and Prakash et al., (2013) in rice.
 

       
On pooled basis, significantly maximum root shoot ratio on dry weight basis (0.350) recorded in the treatment NAA 50 mg/L (T₅) while, minimum (0.187) was obtained by the treatment 2% CaCl₂ (T₁). While, the treatment T₅ remained at par with the treatment NAA 25 mg/L (T₄) (0.344). The root : shoot ratio is one measure to help you assess the overall health of the plants. Consistent with shoot response to above-ground conditions, root biomass is influenced by below-ground conditions where low availability of either water or nutrients commonly leads to greater root : shoot ratio. These results are in accordance with Patil (1987) in sorghum and Rangaswamy et al., (1993) in red gram, sorghum, groundnut and cowpea.
       
Though the treatment differences were non significant, the treatment Cycocel 1000 mg/L (T₃) recorded numerically highest root shoot ratio on length basis (1.399 and 1.406) followed by the treatments 2% CaCl₂ (T₁) (1.395 and 1.400) and Cycocel 500 mg/L (T₂) (1.329 and 1.348) during the year 2015-16 and 2016-17, respectively. The minimum root shoot ratio observed in the treatment NAA 50 mg/L (T₅) (1.084 and 1.079 respectively). But over the years, significant differences observed on pooled basis. The seed hardening treatment Cycocel 1000 mg/L (T₃) recorded significantly superior root shoot ratio (1.402) and remained at par with Cycocel 500 mg/L (T₂) (1.399) and 2% CaCl₂ (T₁) (1.398), while minimum value (1.081) observed in NAA 50 mg/L (T₅).

On the basis of above finding it can be concluded that pre sowing seed hardening treatment with 2% CaCl₂ and other chemicals played an effective role in improving seedling quality and morpho-physiological characters in green gram. Seed germination and initial growth enhanced which helps in better establishment of seedling during early growth. Improvement of seed quality by seed hardening with 2% CaCl₂ is a simple and easy approach to enhance the germination, vigour, root shoot ratio, dry weight and overall seed performance and thereby agricultural productivity especially in the dry land and marginal lands of resource poor farmers.