Water Stress Induced Changes in Seed quality of Fenugreek (Trigonella foenum- graecum L.) Genotypes

Fenugreek (Trigonella foenum-graecum L.) 2n = 16 (Fryer, 1930) popularly known by its vernacular name ‘methi’ is an important condiment crop largely grown in northern India during Rabi season. Fenugreek occupies a prime position among various seed spices grown in India. It is an annual herb belonging to sub-family papilionaceae of the family leguminaceae. It is mainly used as a condiment, but its seeds are also used as carminative and are an ingredient of several ayurvadic medicines, mainly those prescribed for promoting appetite, correcting disorder and for relieving the pain of joints particularly in old age.  Its tender leaves are consumed as leafy vegetables; chopped leaves are mixed in flour to prepare ‘Parantha’. Seeds have carminative property and are also an important ingredient in concentrate feed for animals.
 
Fenugreek is to believe to be native of South-Eastern Europe and Western Asia and mainly cultivated in India, Argentina, Egypt, Morocco, Southern France, Algeria (Bose 1993). In India it is mainly cultivated in Rajasthan, Gujrat, Tamil Nadu andhra Pradesh, Uttar Pradesh, Himachal Pradesh and Haryana with total area of 12.17 Mha and production of 18.85 M Tonns (Spice Board, India). Fenugreek, however, can be grown with limited water, but irrigation at suitable growth stages resulted an increase in yield and yield contributing traits which showed significant genetic variation (Kumar et al., 2000; Chhibba et al., 2000; Acharya et al., 2006; Jain et al., 2013). One of the most important environmental factors that affect this crop is the availability of water which is included in all vital activities. Due to insufficient, untimely and erratic rainfall in semi-arid and arid areas, the fenugreek crop often suffers from terminal drought. In India, major cultivated area of fenugreek is still dependent on conserved moisture and moisture stress conditions reduced its productivity. Water plays an important role in physiological and biochemical process in plants, hence is major limiting factor for agriculture productivity. Saxena et al., (2017a) reported that good amount of seed yield can be obtained under limited moisture conditions without sacrificing its quality. In fact seed quality is found to increase in under limited moisture stress at flowering stage of crop growth. Seed quality of fenugreek is determined by its primary metabolites and an alkaloid diosgenin. Apart from these fenugreek seeds contains saponins, cellulose, hemicellulose and major nutrients such as calcium, iron, sodium and amino acid like leucine, valine, lysine and phenylalanine with unusual amino acid 4-hydroxyisoleucin. Seed are bitter in taste due to presence of an alkaloid “trigonelline”. In recent years the importance of fenugreek has further increased due to presence of a steroid called “diosgenin”. Diosgenin is used in the synthesis of sex hormone and oral contraceptives ( Rao and Sharma, 1987).

In a recent publication, Rathore et al., (2017) analyzed genotypic variability in fenugreek varieties for seed oil fatty acid composition and observed good combination of n-6 and n-3 fatty acids and recommend for blending of fenugreek oil for obtaining good quality cooking oil. With this preliminary information, present study was undertaken to evaluate promising diverse genotypes from across India for yield, diosgenin content and other grain quality parameters under normal and limited moisture stress conditions.
 

Plant materials
 
The present investigation was carried out with ten fenugreek genotypes namely RMt-1, RMt-143, RMt-303, RMt-305, RMt-351, RMt-354, RMt-361, RMt-365, Hisar Sonali and Rajendra Kanti. Pure seeds of these genotypes were procured from, AICRP on spices, Department of Plant Breeding and Genetics, SKN College of Agriculture, Jobner. A field experiment was carried out at Agronomy farm, S.K.N. Collage of Agriculture, Jobner, Jaipur (Rajasthan) during Rabi 2016-2017. The cite of experiment  situated at latitude of 26005’ N, longitude of 75020’E and at the height of  427 m above mean sea level (Shivran et al., 2016) In order to achieve the objectives of present investigation the experiment was planned and executed as described below:
 
Preparation of samples
 
The present study was conducted in randomized block design (RBD) with three replications. The genotypes were sown on 19 November 2016 using dibbler at a depth of 3-5 cm. The experimental planted in 3m x 2m plot using the spacing between row to row was kept to 30 cm and plant to plant was 10 cm. The recommended dose of fertilizers (20 kg N, 25 kg P₂O₅ and 40 kg K₂O) were applied as per standard agronomic practices. The crop was irrigated regularly to maintain plant growth and development till the moisture stress created. Stress was created in one set of genotypes by alternate irrigation. Seed samples from both non stressed and water stressed plant were used for quality analysis in laboratory. Seed yield and quality parameters were analyzed.
 
All the chemicals used for analysis and biochemical work was of Analytical R grade. All glassware’s used were of either corning or borosil. Analysis of different parameters was done by using standard methods and protocol as per following.
 
Total oil (%)
 
Twenty gram seeds of fenugreek genotypes were ground and oil was extracted with 150.0 ml of n-hexane following the procedure of Soxhlet (1879) extraction:
   
Saponin content (%)
 
The defatted cake (residue after oil extraction) was kept under the hood overnight. The next day 100 ml methanol was added to the cake and the mixture was shacked overnight on a rotary shaker at 50 rpm. This mixture was centrifuged at 4000 rpm for 5 minutes. The centrifugation process was repeated three times and supernatant were pooled. Methanol was evaporated in a hood to obtained yellow crystal powder of crude sapogenins.
 
Diosgenin content (%)
 
Diosgenin was determined as per the methods described by Baccau et al., (1977) and Uematsu et al., (2000), with some modification. Standard Diosgenine and p-Anisaldehyde were procured from sigma-aldrich, USA. All other chemicals were of analytical grade. The diosgenin level was determined by measuring absorbance at 430 nm, based on the colour reaction with anisaldehyde, sulphuric acid and ethyl acetate. Briefly, two colour developing reagent solutions were prepared: (A) 0.5 ml p-Anisaldehyde (99%) and 99.5 ml ethyl acetate and (B) 50 ml concentrated sulphuric acid and 50 ml ethyl acetate. A 1000 ppm solution of crude saponins was prepared in methanol. Hundred milli litre of this solution was placed in another glass tube. The methanol was evaporated under reduced pressure. This residue was dissolved in 2 ml of ethyl acetate; 1 ml each of reagents A and B were added to the tube and stirred. The test tube was placed in a water bath maintained at 60°C for 10 minutes to develop colour and then allowed to cool down for 10 min in 25°C. The absorbance of the developed colour was measured with a spectrophotometer (Systronics, type no 2203) at 430 nm. Ethyl acetate was used as a control for the measurement of absorbance. As a reagent blank, 2 ml ethyl acetate was placed in a tube and assayed in similar manner. For the calibration curve, 10-80 ppm standard diosgenin in 2 ml ethyl acetate was used. Each sample was repeated thrice and the average was taken. The amount of diosgenin was calculated by using the standard curve of diosgenin prepared with methanol.
 
Moisture percent
 
Moisture content was determined using oven dry method (Mehta and Lodha, 1979).
 
Crude protein (%)
 
Percent nitrogen was estimated by digesting the samples with sulphuric acid using hydrogen peroxide to remove black colour. Estimation of nitrogen was done by colorimetric method using Nessle` s reagent to develop colour (Snell and Snell, 1939). Protein content in grains was calculated by multiplying nitrogen content (%) by the factor 6.25 (AOAC, 1995).
 
Total Soluble Sugar (%)
 
Hundred milligram of the fenugreek seed powder was extracted with 5 ml of boiling 80% ethanol. Solution was centrifuged at 5000 rpm for 10 min using a table top centrifuge machine. Supernatant was collected in a small beaker and evaporate it on a hot plate using fume hood. The residue was dissolved in 10 ml dH₂O. Pipette out 0.2 ml of the sample extract and make up the volume to 1.0 ml with dH₂O. Four ml of anthrone reagent was added in each test tube slowly and mix carefully. Test tubes were place inboiling water bath for 10.0 min. Take out the test tubes and let them come to room temperature. Optical density (O. D.) of blue green colour of cooled sample was recorded at 620 ηm. Quantity of carbohydrate was expressed as mg/g of the sample.
 
Ash content (%)
 
Ash Content was determined as described in the AOAC (1995) and calculated as percent according to below formula:
  Test weight (g)
 
The sample of 1000 grain was taken from each plot and their weight was recorded using an electronic balance.
 
Seed yield per plant
 
For seed yield five plants were selected randomly of each genotypes and yield was calculated in seed yield per plant (g).
 
Statistical analysis
 
All the observation was taken in each genotypes, replications and sets. The data was statistically analysed according to Panse and Sukhatme, (1985).

Fenugreek crop responds to water stress in the form of changes in various physiological, biochemical and molecular processes. Research on water stress tolerance mechanisms in fenugreek has gained momentum in many laboratories around the world.  In the present study, with ten genotypes varying in quality performance in response to limited irrigation, quality indices have been monitored at two environment conditions, i.e., normal irrigation and limited irrigation. Biochemical parameters were measured for quality evaluation (viz., Total oil, Saponin, Diosgenin, Crude protein, Total soluble sugar, Moisture percent, Ash content. Beside these yield parameters were also measured i.e. test weight and seed yield per plant. All these parameters helped in assessing the quality of fenugreek genotypes under water stress condition.
 
The values for total oil in the fenugreek genotypes revealed that there was increase in total oil percent under moisture stress in all genotypes. The total oil varied from 2.68% (RMt-305) to 3.95% (RMt-1) under non stress conditions, while under moisture stress it varied from 3.43% (RMt-351) to 4.34% (RMt-1). Thus, study showed significant increase in total oil content due to water stress. The maximum increase was observed in genotype RMt-305 (33.42%) followed by RMt-351 (22.62%) and RMt-303 (15.05%) presented in Table 1.


 
Effect of moisture deficiency on oil content in seeds has been studied also by earlier researchers. General observation is that seed oil reduced in most of the oil seed crops when moisture stress imposed at later stage of plant development (Ali et al., 2009; Dwivedi et al., 1996). However, contrasting observations were made by Conkerton et al., (1989). They reported non-significant effect on seed oil of groundnut varieties on account of moisture deficiency at either early or late growth stage. Similarly, Al-Barrak (2006) and Zarei et al., (2010) also reported no effect of irrigation regimes on seed oil content. In present investigation, we found slightly increased seed oil content when moisture stress applied and provide normal irrigation. Similar results were found by Saxena et al., (2017b). They reported that water stress at mid term growth stage, increased the oil content from a minimum of 3.29% to maximum of 5.31%. In our study, water stress observed increase in total oil content in all the genotypes. Water stress at different growth stages of plant might alter the seed composition and related quality (Anwar et al., 2006).

The saponin content varied from 4.68% (RMt-305) to 5.59% (RMt-361) under non stress, while under limited moisture stress it varied from 5.08% (RMt-305) to 5.80% (RMt-361). The maximum increase was observed in genotype RMt-351 (18.64%) followed by RMt-143 (14.11%) and RMt-305 (8.61%). The results showed significant increase in saponins content due to limited moisture stress (Table 1).
 
Fenugreek is a rich source of steroids such as yamogenine, tigogenin and diosgenin. Diosgenin is the most impartment out of all four mentioned secondary metabolites. Total saponin and diosgenin content in seeds of fenugreek genotypes grown under water stress are presented in (Table 1). The Diosgenin content varied from 0.62% (RMt-354) to 0.84% (RMt-1) under non stress, while under water stress it varied from 0.66% (RMt-354) to 0.91% (RMt-1). Thus the present investigation showed significant increase in Diosgenin content due to limited irrigation. The maximum increase due to limited moisture stress was observed in genotype RMt-143 (14.71%) followed by Hisar Sonali (10.23%) and Rajendra Kanti (9.76%). On the basis of increase in Diosgenin under moisture stress, genotype RMt-143 seems to be tolerant to water stress (Table 1). Several researchers evaluated fenugreek germplasm for sapogenin and diosgenin content and reported 0.28-0.92% diosgenin (Taylor et al., 2002) and 0.92-1.68% steroidal saponins (Arivalagan et al., 2013) in mature seeds of fenugreek which is similar to present report.
 
In the present study the moisture percent was found to decrease, in all the genotypes during water stress. The values of moisture percent varied from 5.51% (RMt-354) to 5.93% (RMt-365) under non stress control, while under water stress it varied from 4.42% (Rajendra Kanti) to 4.79% (RMt-361). The minimum decrease due to water stress was observed in genotype RMt-305 (13.75%) followed by RMt-143 (16.26%) and RMt-354 (17.26%). Results of present investigation showed significant reduction in seed moisture content under water stress condition (Table 2). The moisture percent in the seeds is of utmost importance, if the moisture % is higher to a certain level it may creak the fungus effect on the seed, while on storage i.e. lower the percent better for quality point of view. Genotype Rajendra Kanti has lowest moisture % in limited moisture stress while higher reduction in moisture % was noticed in genotype RMt-365 (23.33%).


 
Seed crude protein varied from 23.75% (RMt-365) to 27.56% (Hisar Sonali) under non stress control, which decreased slightly when water stress was applied to 22.94% (RMt-365) to 26.98 % (Hisar Sonali). The minimum decrease was, however, observed in genotype RMt-351 (1.44%) followed by RMt-1 (1.53%) and Hisar Sonali (2.10%). Table 2. The fenugreek seeds were the richest source of crude protein (Kochhar et al., 2006). Researchers (Gopalan et al., 1992; Saibaba and Raghuram, 1997) have reported that fenugreek seeds are rich in protein with a well-balanced amino acid pattern. This could probably be attributed to the increase in N₂-fixing efficiency of inoculated plants where more nitrogen was fixed and translocated to the seed. There are reports indicating decrease in crude protein content in seeds of lentil under water stress (Sehgal et al., 2019).

The total soluble sugar was also found to reduce under moisture stress. It was recorded 4.44% (Rajendra Kanti) to 5.86% (RMt-365) under non stress and reduced up to 3.80% (RMt-351) to 5.66% (Hisar Sonali) under water stress conditions. Minimum decrease was observed in genotypes Hisar Sonali (1.62%) followed by RMt-361 (3.47%) and RMt-365 (4.21%) (Table 2). Similar findings were also observed by Gopalan et al., (1992); Kochhar et al., (2006); Saibaba and Raghuram (1997) and Sumayya et al., (2012).
 
Ash content is the digestion of mineral matter had significant variation among genotypes. Water stress was, however, found to be reduced in all the genotypes. The ash percent varied from 3.54% (RMt-303) to 4.59% (RMt-351) under non stress conditions, while under moisture stress it varied from 3.10% (RMt-1) to 4.29% (RMt-354). Amongst the ten genotypes, the lowest reduction was observed in genotypes RMt-354 (1.05%) followed by RMt-143 (2.63%) and RMt-303 (3.09%) due to water stress (Fig 1). The presence of ash in fenugreek plant in such quantities are satisfying, because of the high importance of mineral for health maintenance and development. Ash consider basic element of biomolecules (protein, enzyme, phospholipid) in addition to their roles in connectivity process and in all of biochemical reaction (Jim, and Stewart, 2002). Our results are consistent with the findings in lentil crop Sehgal et al., (2019).


 
Test weight (g) ranged from a minimum of 11.01g (Rajendra Kanti) to the maximum of 14.44 g (RMt-361) under non stress control while under water stress it reduced and ranged from a minimum of 10.69g (Rajendra Kanti) to the maximum of 13.94g (RMt-361). All the genotypes showed significant reduction in test weight under moisture stress conditions. However, minimum decrease was observed in genotype RMt-365 (2.65%) followed by Rajendra Kanti (2.92%) and RMt-303 (3.05%). The findings are in consonance with Basu et al., (2009). Test weight of seeds is directly proportional to seed moisture content. In present investigation seed moisture content was also found to reduce under water stress conditions resulted in less test weight (Fig 2).


 
A significant reduction in seed yield was recorded in response to water stress irrespective of the genotypes. It was ranged from 2.78 g per plant (Rajendra Kanti) to 4.56 g per plant (RMt-361) under stress as compared to 3.92 g per plant (Rajendra Kanti) to 5.67g per plant (RMt-361) under non stress conditions.  Genotype RMt-1 had least reduction in yield under water stress condition (3.54%) followed by RMt-365 (10.34%) and RMt-303 (17.29%) with a maximum reduction in genotype Rajendra Kanti (29.19%) indicating that this genotype is most susceptible to moisture stress conditions (Fig 3). In an earlier report, Ahari et al., (2009) found varietal difference in drought resistance of fenugreek. The highest phenotypic and genotypic variances are found in seed yield under rain fed conditions and plant type and growth habit in both rains fed and irrigated conditions. Hussein and Zaki, (2013) reported that the missing of 4^th irrigation in fenugreek decreased fresh and dry weight by 52.96 and 47.3% as compared with control plants. Thus it can be concluded that, an extensive water stress tolerance mechanism is active in fenugreek crop. Drought susceptibility of a genotype is often measured as a function of the reduction in yield under drought stress (Blum, 1988).  In the present study, there is reduction in yield due to water stress in all the genotypes studied. Genotype RMt-1 was less affected in limited irrigation. The findings are in consonance with Meena et al., (2016) and Giridhar et al., (2016).

Underutilized crops like fenugreek should be seen as an alternative for crop diversification in the areas of limited water availability for satisfying nutritional requirement of increasing population. The results of present investigations indicated significant effect of water stress on quality parameters where an increase was observed in total oil, saponin content  and diosgenin content  while decreases in crude protein, TSS content, moisture percent, ash content, mineral composition, test weight and seed yield. Genotype RMt-1 accumulates high total oil and diosgenine content while the genotype RMt-1 accumulates high total oil and diosgenine content under both the conditions. Hisar sonali was least affected for crude protein and total soluble sugar under stress conditions. Study revealed that fenugreek can be grown with limited moisture by judicious application of available water without affecting quality attributes viz., total oil, diosgenin content and saponin content; rather, it increases under moisture stress. Fenugreek genotypes like RMt-361 and RMt-1, are rich in steroidal saponin, diosgenin, could be utilized as alternative sources for the synthesis of steroid drugs in pharmaceutical industries.