Effect of organic seed pelleting on seedling quality, gas exchange, growth, yield and resultant seed quality parameters of black gram

In India, black gram is grown in 3.10 million hectare area with a production of 1.40 million tonnes. India  is  the  major  producer  and  consumer  of  pulses  in  the  world  accounting  for  35  and  25  per cent of  the  world’s  area  and  production  under  pulses  respectively (Tiwari and Shivhare, 2016). As a restorative crop of soil fertility, pulses has a unique position in cropping systems of wet land, dry land or rainfed agriculture. The heavy leaf drop increases the organic matter in soil and many of pulses can also fix large amount of nitrogen through symbiosis and thus minimize the dependency on chemical fertilizers. The pulses also play a vital role in providing protein rich food to human beings and in sustaining both soil health and crop production on long-term basis. Though pulses are protein rich crops,but they are still being cultivated in more than 78 per cent of the energy starved condition. Hence, the level of productivity of these crops in India is far below the average productivity of the world
        
Of late in India, the average productivity of black gram crop has rather remained static (343 kg ha^-1) due to several reasons.  Apart from the genetic makeup, the physiological factors viz., poor germination, insufficient partioning of assimilates, poor pod setting due to the flower abscission and lack of nutrients during critical stages of crop growth, coupled with a number of diseases and  pests were the reasons for the poor yield. Several strategies have been initiated to boost the productivity of black gram. One among them is seed pelleting with  botanicals which will give an initial  boost  for  germinating the seeds  and  growing  seedlings which can put  forth  better  root  and  shoot  growth  and  thereby enhance  the  drought  tolerance  resulting  in  increased  yield. The cost involved in seed pelleting is less but the benefit to the farmers is more.
        
Many researchers have proved the use of these leaf powders in enhancing the crop yield in many crops. Bhattacharya et al., (2015) studied seed invigouration treatments in soybean and found that pre storage dry treatments with ferulic acid, aspirin and para-amino benzoic acid  improved storability and field performance of stored soybean seed. The vigour of the seedling is an important factor in crop production and it is generally believed that large sized seeds would give vigorous seedlings than small  and ungraded seeds (Mandal et al., 2000). Mandal et al., (2000) and Sengupta et al., (2005) reported that beneficial effects of dry seed vigoration treatments in several other crops and suggested that counteraction of free radical reaction and lipid peroxidation may be the possible cause of  viability maintenance and good emergence. The anti oxidant property of pungam leaf extract has been well documented by Himani Pande et al., (2014) and Rahul Deo Yadav et al., (2011).
        
Hence seed pelleting with botanicals has gained importance nowadays. Considering the above facts, experiments were undertaken to study the effect of botanical seed pelleting on morpho physiological characters, leaf chlorophyll, gas exchange, growth, yield and resultant seed quality parameters in black gram variety CO 6. 

Laboratory and field experiments and subsequent analysis were carried out at the Experimental Farm and Seed Technology Laboratory respectively in the Department of Genetics and Plant Breeding, Faculty of Agriculture, Annamalai University, Tamil Nadu, India during 2016 - 2017.  The field is situated at 11° 24' North latitude and 79° 69' East longitude at an altitude of + 5.79 m above mean sea level. The soil of the experimental field was clayey loam. Genetically pure seeds of black gram [Vigna mungo (L.) Hepper] cv. CO 6 obtained from Tamil Nadu Agricultural University, Coimbatore was selected for the study.
        
pelleting is one of the methods which results in modifying the physiological and biological nature of seed that are favourable for direct sown condition. Pelleted seeds perform better than that of the unpelleted seeds due to their influence in enriching the efficiency of rhizosphere soil for production of quality seedlings. Seed pelleting is a one of a management practice/tool which alone or in combination with other practices could bring about a reasonably good degree of reduction of inoculum potential and at the same time ensure the sustainability of the production, cost effectiveness and healthy ecosystem. The following process of seed pelleting with different kinds of pelleting materials should be followed.
 

Process of pelleting               

 

                                               

The seeds are uniformly coated with adhesive in correct quantity initially. Then the filler materials are sprinkled on the coated seeds and are rolled on the filler material for effective and uniform coating. The thickness of the seed coating is dependent on the amount of sticker in relation to the amount of seed. The total mixing time should not exceed four minutes since prolonged agitation may damage the seeds or chips of pelleted coat. The bulk seeds of black gram [Vigna mungo (L.) Hepper] cv. CO 6 were graded for uniformity and then imposed to various pelleting treatments with leaf powders of two botanicals viz., pungam (Pongamia glabra)  and prosopis (Prosopis juliflora) was done by using gum Arabica as adhesive to study the effect of botanical seed pelleting on seedling quality, biometric,  gas exchange and yield parameters in the laboratory and field with the following treatments.
 
T₀    -    Control
T₁    -    Seed pelleting with pungam leaf powder @ 50g kg^-1 
T₂    -    Seed pelleting with pungam leaf powder @ 100g kg^-1
T₃    -    Seed pelleting with pungam leaf powder @ 150g kg^-1
T₄    -    Seed pelleting with pungam leaf powder @ 200g kg^-1
T₅    -    Seed pelleting with prosopis leaf powder @ 50g kg^-1
T₆    -    Seed pelleting with prosopis leaf powder @ 100g kg^-1
T₇    -    Seed pelleting with prosopis leaf powder @ 150g kg^-1
T₈    -    Seed pelleting with prosopis leaf powder @ 200g kg^-1
 
Laboratory analysis
 
The pelleted seeds were dried under shade for two days and were evaluated for the morpho physiological characters. The germination test was conducted as per the method of ISTA (1999). Germination test was conducted with four replicates of hundred seeds each by following between paper method in the germination room, maintained at 25 ± 1°C and 96 ± 2 per cent RH. The germination % was calculated based on the normal seedling count on 10th day and it was expressed in percentage. Germination percentage was calculated by adopting the formula of Magurie (1962) and (ISTA, 1999).
 
Ten normal seedlings were taken at the end of the germination test for measuring the root length. The portion from the collar region to the tip of the root was measured in cm and the mean value was recorded and expressed in cm. The same seedlings used for root length measurement, were used for shoot length measurement. The length from the collar region to the tip of the first leaf was measured and the mean value was recorded and expressed in cm.
       
The seedlings used for growth measurement were placed in a paper cover and dried in shade for 24 h and then, the paper cover along with seedlings were kept in an oven maintained at 85° ± 1°C for 24 h. The dried seedlings were cooled using desiccators for 30 min. The dried seedlings were weighed and then mean value was expressed in mg seedling^-10. The vigour index of the seedling was computed using the following formula suggested by Abdul-Baki and Anderson (1973) and expressed in whole number.  

 
Vigour index  =  Germination (per cent)x Dry matter production

 
Field evaluation
 
Field trial was conducted by adopting randomized block design with three replications under dry land condition. The plot size was 4×2.5 m². The crop was raised with the spacing of 30 × 10 cm and recommended package of practices were followed. Proper irrigation was done at critical stages of  flowering and pod formation and observations on growth, gas exchange and yield parameters were recorded.
        
Plant height was recorded by measuring the height of plant from ground level to the tip of main branch using a meter scale and the mean value was expressed in centimeter. Total number of branches plant^-1 was counted and the mean number of branches plant^-1 was recorded as whole number. The biomass production was recorded from ten seedlings selected at random separately, uprooted with root system intact and washed to remove the soil particles, placed in a paper cover, shade dried for 24h and then in the hot air oven maintained at 100°C for 24h. The dried plants were cooled in a desiccator for 30 minutes and the mean weight was recorded in grams. Number of days taken from sowing to first flowering at any node of plant in each of the plots was recorded and the mean value is expressed as days to first flowering in whole number. Number of days taken from sowing to 50 per cent flowering in the total population was recorded and the mean value is expressed as days to 50% flowering in whole number.
        
The total chlorophyll was calculated using the formula as suggested by Yoshida et al., (1971) and expressed as mg g^-1. Leaf photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO₂ concentrations were measured from two, uppermost fully expanded leaves on intact plants in the field using LICOR-6400×T Portable Photosynthetic System (Lioncoln, USA). All these estimations and measurements were made between 10.00-11.00. a.m on the five replicates for each treatment.
         
At maturity, the pods obtained in each of the plants were measured for their length using scale. Excluding the stalk, the length was measured from the base to the tip of the pod and the mean pod length was expressed in centimetres. The number of pods plant-1 was counted and the mean number of pods plant^-1 was arrived at and expressed as whole number. The pod yield plant^-1 at maturity was recorded by using an electronic balance in randomly selected plants and the mean pod yield plant^-1 was arrived at and expressed in grams. The pods used for recording pod length were split longitudinally and the number of seeds in each pod was counted. Mean number of seeds pod^-1 was calculated and reported as whole number.
          
The pods from ten tagged plants in each treatment were hand shelled. The seeds were cleaned and weighed in an electronic balance and the mean seed yield plant^-1 was expressed in grams. The seeds thus obtained were cleaned and weighed to arrive at the plot yield. Seed yield was computed per hectare from the seed yield obtained per plot in each of the treatment and expressed in kg ha^-1. Eight replicates of 100 seeds were drawn from each treatment randomly, weighed in an electronic balance and the mean weight was expressed as 100 seed weight in grams (ISTA, 1999). All the data were analysed statistically as per the method of Panse and Sukhatme (1985) with appropriate tools and expressed as mean values.

Seedling quality parameters
 
Seeds pelleted with pungam leaf powder @ 150 g kg^-1 recorded higher germination percentage (93.00), root length (18.35 cm), shoot length (19.34 cm), dry matter production (0.48 mg seedling^-10) and vigour index (3505.10), whereas the lowest value was observed in control (Table 1). The reason for higher germination of pungam leaf extract treated seed may be greater hydration of colloids and higher viscosity of protoplasm and cell membrane that allows the early entrance of moisture that activates the early hydrolysis of reserve food materials in the seed as compared to untreated seeds. These biocontents might synergistically interact with aminoacids especially tryptophan to form the indole acetic acid (IAA) in germinating seeds to bring about enhancement in seedling growth (Lu et al., 1983). Pungam leaf powder contains mineral nutrients like nitrogen (5.6%), phosphorus (P₂0₅-0.9%), potassium (K₂ 0-3.l1%) and calcium (Ca 0-1.0%) and GA₃ in traces (Nadeem Binzia, 1992). The higher germination might also be due to the role of calcium as an enzyme cofactor in germination process by increasing protein synthesis as reported by Christansen and Foy (1979). Similar observations were made by Maheswari (1996) and Yadav et al., (2014). The increased shoot length and root length in seeds treated with botanical leaf powder may be attributed to cell wall extension and increased metabolic activities (Afzal et al., 2002). The finding of Ramesh Kumar and Muthukrishnan (2015) also corroborated with the results of present study. The pronounced increase in seedling length and dry matter production of pungam pelleted seeds may be due to activation of the growth promoting substances and translocation of secondary metabolites to the growing seedling (Napar et al., 2012).
 

 
The increase in dry weight was claimed to be due to enhanced lipid utilization and enzyme activity due to the presence of bioactive substances like auxin in pungam leaf extract (Rathinavel and Dharmalingam, 1999) and development of seedling to reach autotropic stage and enabling them to produce relatively more quantity of dry matter with hike in vigour index by pelleting treatment. Similar results were reported by Tamilmani (2012) in black gram and Prakash et al., (2013) in rice. The increase in dry weight with botanicals treatment may be due to the faster growth and development of seedling and hike in vigour index (Sathiya Narayanan et al., 2016). Benefits of seed treatment with prosopis and pungam leaf extracts has already been reported by Khan Bahadar Marwat and Muhammed Azim khan (2006) in wheat and Renugadevi et al., (2008) in cluster bean. Physiologically active substances may have activated the embryo and other associated structures which results in absorption of more water due to cell wall elasticity and development of stronger and efficient root system which leads to the increase of higher vigour index (Basra et al., 2005). Similar observation was made by Anbarasan et al., (2016) in red gram.
 
Growth parameters
 
From the present investigation, it was observed that the seeds pelleted with pungam leaf powder @ 150 g kg^-1 recorded higher values for growth parameters like plant height (48.73 cm), number of branches (8.70), number of nodules plant^-1 (12.00), dry matter production (15.00 g plant^-1) and lower days to first flowering (23.23) and 50 percent flowering (30.12) whereas control treatment has recorded lower values for all the above growth parameters (Table 2).  Plant height is very important criterion for a crop in providing more places for flower production leading to better yield. Pungam leaf extract hardening improved the growth attributes by triggered biosynthesis of nucleic acid, proteins, hydrolytic enzymes and consequentially enhanced the cell division, cell enlargement, metabolic activity and increased the photosynthetic process of the plant resulting in increased uptake of more nutrients by efficient and stronger roots. The initial vigour of the pungam leaf powder invigourated seeds might have induced the seedling growth and enabled better nutrient absorption by the foliage, thus encouraging quick growth and increased plant height with increased number of branches (Dileepkumar et al., 2009). The increased number of branches and plant height could be attributed to the presence of ammoniacal nitrogen, phosphorous and other essential micronutrients in the pelleting combination. The improvement in field emergence could be attributed to activation of cells, which resulted in the enhancement of mitochondrial activity leading to the formation of more high energy compounds and vital biomolecules, which are made available during the early phase of germination (Ananthi et al., 2015).
 

 
Leaf chlorophyll content
 
Among the  seed pelleting  treatments,  pungam leaf powder pelleted seeds @ 150 g kg^-1 recorded higher chlorophyll content ‘a’ (0.94 mg g^-1) and was followed by seeds pelleted with prosopis leaf powder @ 50 g kg^-1 (0.84 mg g^-1) and untreated seeds (T0) which recorded lowest chlorophyll ‘a’ content  (0.63 mg g^-1) (Table 3). Similar observations were recorded for chlorophyll ‘b’ and total chlorophyll contents with pungam leaf powder pelleted seeds @ 150 g kg^-1 followed by the seeds pelleted with prosopis leaf powder @ 50 g kg^-1.  Increased chlorophyll ‘a’, ‘b’ and total chlorophyll observed in pungam leaf powder pelleting @ 150 g kg^-1 might be due to the maximum production of chemical energy and plant metabolism which results in plant growth (Ayumi et al., 2004). Because of the invigorative effect of prosopis, the plants would have absorbed more nutrients from the soil and utilized for more chlorophyll production resulting in enhanced photosynthetic activity of treated plants (Sathiya Narayanan et al., 2015). This increase could be also due to the presence of mineral nutrients like nitrogen, potassium and calcium which plays a major role in chlorophyll synthesis (Prakash et al., 2013). Mahmood et al., (2007) observed increased chlorophyll content due to application of organic matters. Similarly, increased chlorophyll content with botanicals such as Argemone mexicana, Calotropis procera, Solanum xanthocarpum and Eichhornia echinulata were also reported by Rizvi et al., (2012). 
 

 
Leaf gas exchange parameters
 
Photosynthesis is one of the most vital physiological processes contributing to plant growth and productivity by enhancing dry matter. From the results, it was found that plants treated with pungam leaf powder @ 150 g kg^-1 recorded more photosynthesis and transpiration rates (25.92 mg CO₂ m^-2s^-1 and 10.62 mg H₂ O m_-2s_-1) followed by seeds pelleted with prosopis leaf powder treatment @ 50 g kg^-1 (25.83 mg CO₂ m^-2s^-1 and 10.30 mg H₂ O m^-2s^-1) and the lowest values were recorded in untreated seeds (Table 3). For the intercellular CO₂ concentration and stomatal conductance, higher values were recorded by plants treated with pungam leaf powder pelleted seeds @ 150 g kg^-1 (274.40 µ mol mol^-1 and  0.80 mol m^-2s^-1)  followed by seeds pelleted with prosopis leaf powder @ 50 g kg^-1 (272.013 µ  mol mol^-1 and  0.76 mol  m^-2s^-1). Increase in gas exchange parameters might be due to increased enzyme activity, presence of growth promoting substances like GA₃ and increase in chlorophyll content that leads to more production of photo-assimilates and better growth and development. Similar results of increased gas exchange parameters was already reported by Prakash et al., (2013) and Sathiya Narayanan et al., (2016). Increased chlorophyll index and improved photosynthetic efficiency of the plant was reported in Ocimum sanctum (Anbarasan et al., 2016).
 
Yield parameters
 
All the yield parameters were found higher in pungam leaf powder pelleting treatment @ 150 g kg^-1, when compared to the other treatments and control (Table 4). The pungam leaf powder pelleted seeds @ 150 g kg^-1 recorded  highest pod length (5.60 cm), more number of pods plant^-1 (26.30),  maximum pod yield plant^-1 (7.30g), more number of seeds pod^-1 (6.50), higher seed yield plant^-1 (9.30 g), seed yield ha^-1 (787.3 kg) and 100 seed weight (6.12 g) and the lowest values were observed in untreated seeds. Physiologically active substances might have activated the embryo and other associated structures which resulted in the absorption of more water due to cell wall elasticity and development of stronger and efficient root system which in turn might have favoured derivation of more nutrients thus enabling better growth resulting in higher yield. Increase in pod yield may be due to the translocation of sugars from complex compounds like carbohydrates and translocated at greater case (Dileepkumar et al., 2009).
 

        
The increase in number of seeds may be due to increased pollen production and enhanced fertilization resulting in increased number of filled seeds.  The leaves of Pongmia pinnata contain 1.16% Nitrogen, 0.14% Phosphorus and 0.49% Potash (Singh,1982) and various alkaloids like pinnalin, pongamol, Saponin, b-sitosterol and tannins (Savita Sangwan et al., 2010). Saponins present in the pongamia leaf extract might have enhanced the nutrient absorption and also would have protected the seedlings against pathogens (Manisathiya and Muthuchelian, 2010). The pungam leaf powder pelleting might have triggered enhanced protein synthesis, which in turn could have contributed to the amino acid rescue and protein turnover during active metabolism, later in plant life. Increase in yield parameters of pelleted seeds may be due to the presence of auxin like substances in leaf extract which regulates the growth and initial establishment of plant. Seed yield in pungum leaf powder pelleted seeds may be due to the increased activity of dehydrogenase, amylase and peroxidase enzymes by the presence of growth regulators like GA₃ (Shehzad et al., 2012). The increase in yield parameters might be due to the bioactive chemicals present in the pungam leaf powder which triggers the synthesis of gibberellin that improves the germination percentage by quickening the germination process which leads to increase in crop growth and development and improves the yield. Similar observations with increased yield parameters were reported by Prakash et al., (2013). Increased yield and yield parameters with flyash seed pelleting has already been reported in black gram (Prakash et al., 2012), bhendi (Prakash et al., 2014a), sesame (Prakash et al., 2014b) and  rice (Prakash et al., 2014c).

To conclude seeds pelleted with pungam leaf powder @ 150 g kg^-1 recorded higher growth and yield parameters such as pod length, number of pods plant^-1 and pod yield plant^-1. Since pulses are grown in rainfed areas which nutrient starved generally, seed pelleting with pungam leaf powder @ 150 g kg^-1 can be recommended for getting assured  yield in black gram.