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Microbe-induced resistance against pathogens and herbivores: evidence of effectiveness in agriculture. In: Agarwal, A.A., Tuzun, S., Bent., E. Eds., Induced Plant Defenses Against Pathogens and Herbivores: Biochemistry, Ecology and Agriculture. APS Press, St Paul, MN, Pp 33. Journal of Food Legumes 263 4: 90-96, 2013 Root morphology and architecture CRIDA indigenous root chamber-pin board method of two morphologically contrasting genotypes of mungbean under varied water conditions V. MARUTHI, K. SRINIVAS, K.S. REDDY, B.M.K. REDDY, B.M.K. RAJU, M. PURUSHOTHAM REDDY, D.G.M. SAROJA and K. SURENDER RAO Central Research Institute for Dryland Agriculture CRIDA, Saidabad post, Hyderabad, Andhra Pradesh, India; E-mail : vmaruthicrida.in Received : June 29, 2013; Accepted : November 18, 2013 ABSTRACT An experiment was conducted in the root chambers during 2010-11 under the net-house conditions at Central Research Institute for Dryland Agriculture CRIDA Hyderabad, India to study the effect of soil moisture deficit on root morphology and root architecture of two morphologically contrasting cultivars of mungbean in comparison with the irrigated treatment. ML267 short stature and WGG37 tall stature were studied with two watering treatments viz., soil moisture up to FC irrigated and another up to 33.3 Available Water Content deficit watered. Soil moisture stress affected total root length at flowering stage 42DAS of mungbean by 30 irrespective of cultivar and also expressed drought resistance of ML267 at vegetative stage 22DAS and at pod filling stage 69DAS of WGG37. Deficit soil moisture conditions affected WGG37 more than ML267. It is concluded that soil moisture deficit conditions 33.3AWC though reduced the root dry weight at the top soil depth, increased Total Root Length TRL, Root Length Density RLD, Root Surface Area RSA at deeper soil depths especially at 42DAS resulting in high root shoot ratio and fine root length of ML267. 2D Root architecture image of a single plant and root measurement at different soil profile depths could be possible in Root chamber-Pin board methodology. Therefore, this indigenized Root chamber- Pin board method was adopted and is recommended for study of root architecture under controlled conditions. Key Words: Mungbean , Root architecture, Root chamber-Pin board methodology, root length density, root morphology, soil moisture stress, total root length. Plants vary in their abilities to survive under extreme environmental conditions. Though visible above ground biomass is the manifestation of the extreme situation being faced, below ground biomass stabilizes the system by playing a crucial role for resilience. Plants capability to proliferate roots and responses to heterogeneous environments and specifically of species and varieties Campbell et al. 1991, Bauerle et al. 2008 differ. With the dynamic nature of soil, changes in root distribution may be observed both horizontally and vertically. Further this is aggravated by the problems of late onset of monsoon, intermittent dryspells droughts during the crop growth period etc. Mungbean Vigna radiate L. R. Wilczek also called ‘Greengram’ is gaining importance due to its contribution to the health of both rich and poor worldwide. Mungbean is a short duration legume of 65-75 days yielding protein rich seeds for human beings, husk and haulms for fodder concurrently improving the soil health through nitrogen fixation. It is generally grown in the rainfed lands of South India, parts of U.P., Madhya Pradesh, Maharashtra and parts of Eastern India. Due to its growing under marginal rainfed situations, production and productivity improvement is not considerable. Therefore efforts are on to break the plateau in the production of these legumes. Generally the first casualty under drought situation is the root system which is an interface between the plant and the soil. Consequently the water saving mechanisms of the plant would come into the scene along with the water capturing abilities of the root system. However, till now the drought management measures were formulated and validated based on the study of only above ground biomass. But the study of below ground biomass and its relation with the yield may help us in finding not only the moisture sensitive stages of the crops accurately and to manage it effectively but also will generate information on critical root traits for extreme situation for plant breeders. Understanding the root distribution soil profile wise in order to estimate the capabilities of the plant to extract soil moisture and nutrients for its sustenance is incomplete without the standard methodologies for root architectural study. Till now the plant root studies mostly were in the form of excavating the total root biomass and estimating the root parameters of the total root system. However, the data did not give much clue about the distribution of roots at different soil depths in the soil profile which primarily affect the acquisition of soil moisture and nutrients. But after long periods of root research, methods like root chamber-pinboard were in vogue since 60s. However, the improvements in this method took place in terms of materials used to fabricate root boxes, methodology to sample etc. Therefore, an attempt was made to indigenize the above mentioned pin board methodology to suit the mungbean root architecture sampling requirements. Mostly these methodologies work with the narrow or thin root boxes which were helpful to get 2D root architecture but Maruthi et. al., : Root morphology and architecture CRIDA indigenous root chamber-pin board method 9 1 to thwart criticism on restricted space for the plant, in India we worked with the objective of single plant field spacing SPFS, by providing boxes of size equivalent to field spacing of a single plant while methodology of extracting root architecture was one another objective being addressed. Moisture within the soil profile is heterogeneous and varies within soil but the root systems must forage for this limited resource. Root system in some cultivars grows vertically with more root angle to the soil surface might have the genetic component Vegapareddy et al. 2010 to exploit more moisture from deeper layers of the soil with the top soil layers dried out Doussan et al. 2003. For the transient soil moisture availabilities and deficits, root system of the plant should be geared up to seek soil moisture for either maintenance of the root under dry soil conditions or for sustaining the roots Eissenstat and Caldwell 1988, Hodge 2004, Kosola and Eissenstat 1994, Eissenstat et al.1999. Therefore an experiment was conducted to assess the effect of soil moisture deficits on rooting variability of two morphologically contrasting mungbean cultivars for their suitability to various field transient soil moisture dynamics. MATERIALS AND METHODS The experiment included two mungbean cultivars grown under limited soil moisture irrigated at 33.3 available water content or 66.7 depletion of AWC and at unlimited water conditions Field Capacity. Of these two mungbean cultivars, ML267 a more popular and short statured while WGG37 from Warangal Research Station of ANGRAU of Andhra Pradesh in India is a tall growing variety. This experiment was conducted under the net-house conditions at Central Research Institute for Dryland Agriculture CRIDA Hyderabad, India during 2010. Experimental details The crop was grown in Red Sandy Loam soil profile filled root chambers of 30x15x45cm dimension. Two seeds were sown in each chamber and thinned to one plant after germination. As per the recommendation, fertilizer was supplied to each chamber. For one set of chambers, irrigation was carried out whenever the soil moisture fell below FC and for another set of chambers, whenever the soil moisture recorded 33.3AWC using surface moisture probe upto 20 cm depth. Each chamber uniformly received fertilizer and water content as per the treatment. The soil with 75 sand, 3 silt and 22 clay has neutral P H 7.2, normal EC 0.16dss -1 , low nitrogen content 171kg ha -1 , medium available phosphorus 17.7 kg ha -1 and high potassium 307 kg ha -1 as the soil was from the long term fallow land . Plants were sampled at 22, 42 and 69 days after sowing DAS. Shoot parameters were recorded as above ground biomass. Spatial configuration of plant roots in soil Root architecture was studied by understanding the distribution of Total Root Length TRL and Root Length Density RLD in the soil profile. Methodology of sampling Illustration for Root Architecture Indigenized Root chamber-Pin board methodology The methodology explained by Price et al. 2002 was Indigenised to suit the size of the root chamber in which the plants were grown. Construction of Root chambers: As our test crop is mungbean, the field spacing of 30 x15cm was taken into account and acrylic chambers of 30cm X 15cm x 15cm rectangle boxes were constructed and three boxes were arranged one above the other so as to have the total depth of 45cm. The boxes were glued with a tape to avoid leakage of water from it. A drainage hole was made to the bottom most boxes. On one side length side of the box, acrylic sheet opens like a door which is fitted with the hasps and hinges. Pin Board: A PVC board with spokes motor bike spokes of 16.5cm length, 3.5mm diameter with a screw at the bottom part fixed at grid lines distance of 2.5 cm in alternate rows and painted black. Sampling Protocol: For sampling, the door of each chamber was opened, fitted with the black pin board by pushing the spokes into the soil until it touches the box and turned it around lifting up the chamber leaving whole soil mass with root system on the pin board. Root washing: Roots were washed by keeping the pin board in the wheel barrow. After washing, the mounted root system on the pin board was placed in the water tray, was photographed after it was allowed to gently align on its own. Photography: By placing the black pin board with root system in the water tray, digital images were generated using high end digital camera at a perpendicularly fixed object distance using boom stand. Root Scanning and image analysis: After sampling and photography, the roots were cut for sub sampling, stored in water of glass bottles in a refrigerator at 4ºC until they were scanned using flat bed scanner of STD 4800 and analyzed TRL cm using “WinRHIZO” Regular 2009c Version. Root scans were stored in tiff format and data were generated in text file, later converted into Excel files for further analysis. After scanning, the roots were dried at 40°C for four days for root dry weights. According to Price et al. 2002 the root boxes made of glass and of 1.5 cm in width and 70cm and 50cm in length and depth respectively, while in our case it is an acrylic box of 30x15x45cm dimension which led to changed pinboard size and the pins. In our experiment, we used the bike spokes of 16.5cm length in place of nails but could not paint them to black. These spokes have got bolts to fix and remove at the bottom. On contrary to the nails, the spokes were of 3.5cm in diameter. Further to take the photographs without spokes 9 2 Journal of Food Legumes 263 4, 2013 obstructing the vision, earbuds painted black were fixed in place of spokes. Statistical Design Complete randomized Block design with one way analysis of ANOVA was employed with four replications Gomez and Gomez 1984 to estimate the treatmental differences. moisture deficit conditions which was 0.184 in ML267 while 0.123 was recorded at field capacity indicating the increased root weights with soil moisture deficits which might have happened either due to suberization of big roots and stimulated proliferation of roots during moderate to severe moisture stress or due to reduced shoot weight. Big roots maximum mean root diameter of 0.10-0.25mm were recorded in ML267 at FC over moisture stressed. Generally wider roots are prominent at reduced soil moisture levels which did not happen in case of ML267. In this context, smaller root diameter in dry soil can be due to greater resistance to penetration which happened in case of ML267 at moisture stressed condition. Therefore, presence of fine roots small root diameter under moisture stress may be one of the drought tolerant traits of this cultivar. Illustration on Methodology of Mungbean Root Architectural Sampling RESULTS AND DISCUSSION Variability in root morphology of cultivar ML267 At field capacity, there was a gradual increase in total root length TRL and root length density RLD of ML267 cultivar up to 42 DAS which was declined later at 69DAS. However, moisture deficits 33.3AWC reduced TRL at all the three stages 22, 42 and 69DAS to an extent of 4, 25 and 42 respectively indicating more root length reduction and sensitivity of the stages for moisture deficits especially at 42 and 69DAS Fig 2.. This indicates the maximum negative effect of moisture stress on this variety at both flowering and pod filling stages compared to the vegetative stage. Maximum root dry weight was observed under well watered conditions while under moisture deficit conditions shoot dry weight was more affected Table 1. Among all the stages, root shoot ratios at 69DAS observed to be more under Maximum RSA 1571 cm 2 was recorded at 42DAS under well watered conditions and lowest was recorded at soil moisture deficit conditions. Soil moisture deficit conditions reduced RSA on an average to an extent of 25 and this reduction was more at 69DAS 45. Variability in root morphology of cultivar WGG37 Unlike ML267, WGG37 recorded maximum TRL and RLD at 69DAS under both water sufficient and deficit conditions. However, the moisture deficits reduced TRL and RLD to a great extent at 22DAS followed by 42 and 69DAS 44, 35 and 19 respectively as the crop duration was progressing emphasizing the moisture susceptibility of this cultivar starting from vegetative stage. Root shoot ratio was more with WGG37 under moisture deficit conditions especially at 22 0.222 and 69DAS 0.254. This might not be only due to greater root weight of the plant but also because of reduced shoot biomass at respective Fig. 1: Effect of water treatments on TRL cm of mungbean cultivars during the crop growth period Maruthi et. al., : Root morphology and architecture CRIDA indigenous root chamber-pin board method 9 3 stages further emphasizing the moisture sensitivity of this cultivar Zhang et al. 2009. Both well watered and moisture stressed WGG37 registered greater mean root diameter both at initial stages 22DAS as well as at later stages 69DAS signifying the presence of bigger roots from the initial stages itself which account for increased root dry weights Eissenstat and Yanai 2002, Waisel and Eshal 2002. As the crop progressed, there was increase in root dry weights both under sufficient and deficit water conditions Table 1. High TRL at 69DAS under both well watered and moisture stressed conditions was the genetic trait of the cultivar.This was the genetic trait of the cultivar as TRL peaked at 69 DAS. As regards RSA at moisture sufficient conditions, maximum was achieved by WGG37 at all the stages while under deficit conditions, it increased with moisture stress at later stages 69DAS which may not be helpful for the crop to achieve better yields as argued by Gooding et al 2005 and Andersson et al. 2005 that growing roots compete with grains for all the resources. However, more RSA at 69DAS 1082cm 2 may be an indication of greater resistance to penetration by root as claimed by Munoz-Romero et al. 2010. Root morphology of ML267 Vs WGG37 Under irrigated conditions, WGG37 recorded maximum total root length 16145cm which was 27.5 more than the total root length registered by ML267 while moisture deficits reduced TRL of WGG37 by 27 which is 3 more than the TRL reduction in ML267. However, Vice Versa was observed with Root Surface Area RSA as more RSA 1571cm 2 registered by ML267 compared to WGG37 1477cm 2 respectively. Mungbean crop in general registered reduced root parameters due to moisture deficits however; this reduction was more in WGG37. Cultivars vary in their ability to counteract drought through root component as the crop growth progresses which may be considered for different stages. Stage-wise discussion was carried out in the next paragraph. ML267 recognized for drought resistance, registered increased trend in almost all the parameters including root dry weights with limited moisture over moisture stressed WGG37 Table 1. However, WGG37 showed increasing trend in root dry weights and RSA with increased age, apart from TRL which may imply more partitioning towards root component at later stages may not be of much help in realizing higher yields as optimal partitioning of drymatter between root and shoot is crucial under conditions of moisture stress Kage et al. 2004 and also the stage at which it happens. ML267 expressed sensitivity to soil moisture deficits after flowering stage onwards through reduced TRL, RLD and RSA. However, WGG37 showed increasing trend in the above mentioned root parameters throughout the crop growth period. Different stages of the crop ML267 Vs WGG3 Irrespective of the cultivar and water treatment, maximum root surface area was recorded at 42DAS flowering stage. Of all the stages and cultivars, crop at 42DAS showed more root surface area and small root diameter compared to all other stages signifying the presence of more fine roots at this stage Table 1. However, regarding TRL, WGG37 registered 18 and 22 more TRL than ML267 at vegetative 22DAS with 1252 cm and at pod filling stages 69DAS with 13146 cm respectively while at flowering stage 42DAS it was 22 less than ML267 16754 cm Fig 2 3. This varied TRL of both Root dry weight gplant DAS Shoot dry weight gplant DAS Root Shoot Ratio weight DAS Mean Root Diameter mm DAS Mean Root Surface Area cm 2 DAS Treatments Yield kg ha -1 22 42 69 22 42 69 22 42 69 22 42 69 22 42 69 ML267 FC 678 0.07 0.49 0.47 0.20 3.45 3.89 0.237 0.163 0.123 0.30 0.29 1.10 118 1571 1132 ML267 at 33.3AWC 462 0.05 0.47 0.31 0.22 2.68 1.73 0.240 0.161 0.184 0.28 0.35 1.02 103 1286 617 WGG37FC 729 0.09 0.45 0.73 0.42 2.94 3.82 0.216 0.153 0.185 0.35 0.33 0.88 201 1477 971 WGG37 at 33.3AWC 338 0.04 0.42 0.66 0.18 2.74 2.60 0.246 0.152 0.232 0.33 0.34 1.01 90 997 1082 SEd ± 18.6 0.013 0.098 0.101 0.012 0.375 0.319 0.030 0.016 0.017 0.003 0.032 0.020 3.14 124 39.5 CD at 5 41 0.029 NS 0.220 0.026 NS 0.696 NS NS 0.37 0.006 0.069 0.043 6.8 271 86 Table 1.Yield, root and shoot parameters of mungbean as affected by the water treatments Figure 2. Root architecture of a ML267 and b WGG37 at 42DAS 9 4 Journal of Food Legumes 263 4, 2013 the cultivars under sufficient soil moisture conditions might be due to the genotypic influence of the cultivars. Further, maximum TRL was achieved at a late stage by WGG37 which is significantly conspicuous as was quoted by Jordan et al. 1979 while high value was observed at 42DAS for ML267 Subba Reddy et al. 1999. This was in conformity with the results obtained by Yantai et al. 2011 as was quoted earlier, that the pulses peak at flowering stage in achieving maximum root component. Further according to Sharma et al. 2007 the biomass allocation to roots reduced from 22 to 12.3 from full bloom stage to harvest. More fine roots and increased TRL sustained ML267 in countering drought effectively. Variability in Root Architecture ML267 Vs WGG37 Root architecture is the study of spatial configuration of roots profile wise. In this experiment, we considered TRL, RLD, RSA Root Surface Area depth wise to explain root architecture of these two genotypes of mungbean both under water sufficient and limited environments. During water sufficient conditions, total root length TRL and root length density RLD in both the genotypes were decreasing as we go deep into the soil since the soil moisture available was sufficient at top soil depth. However, initial soil moisture deficits veg stage increased root length in WGG37 at a soil depth of 15-30cm and further to the deeper depths as we go into the soil profile with the progressing crop growth. This shift of root length and root length density was significant in ML267. Cultivar ML267 when grown under water limited environment Fig 3 4, resulted in more TRL and RLD at deeper depths 15-45cm 42DAS and increased RLD over WGG37 might have enhanced water uptake from deeper layers Zhang et al. 2009 contributing to the drought tolerance of the cultivar. As proved by Songsri et al. 2008 though the surface layers dried out, the small root part in the subsoil might be responsible for major water uptake. Therefore, adaptive spatial root distribution is one of the drought alleviation strategies of the dryland crop plant. Therefore, according to Mannur et al. 2009 root length has some importance with respect to moisture extraction from deeper layers of the soil. Figure 3. Root architecture of ML267 with and without soil moisture at 42DAS Figure 4. Effect of soil moisture treatments on RLD of two mungbean cultivars in the soil profile ML267FC 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 0-15 15-30 30-45 S o il d e p th c m Root Length Density cm cm-3 ML267 33.3AWC 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 0-15 15-30 30-45 S o il d e p th c m Root Length Density cm cm-3 WGG37FC 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 0-15 15-30 30-45 S o il d e p th c m Root Length Density cm cm-3 WGG37 33.3AWC 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 0-15 15-30 30-45 S o il d e p th c m Root Length Density cm cm-3 22DAS 42DAS 69DAS Maruthi et. al., : Root morphology and architecture CRIDA indigenous root chamber-pin board method 9 5 Conspicuous root proliferation in the top soil depth at 69DAS in WGG37 specifies more root partitioning even at later stages which may not be conducive to rapid recovery of crop growth and efficient pod set Turk et al. 1980. Both the genotypes recorded maximum RSA at 42DAS. Root surface area recorded by these two genotypes under well watered and moisture limited environments was same, except that cultivar WGG37 with sufficient irrigation was observed to have more RSA at 42DAS than at later stage 69 DAS while it was vice versa under soil moisture deficit conditions Table 1. Relationship between Root Parameters and Yield At field capacity, WGG37 with its more TRL, RLD at 69DAS, more root dry weight and root shoot ratio with low mean root diameter yielded 729 kg ha -1 which was 7 more than ML267 678 kg ha -1 . However, under soil moisture deficit environments, ML267 performed 462 kg ha -1 better than WGG37 338 kg ha -1 by 27. This might be due to more TRL, RLD with deeper soil depths at 42DAS for soil moisture. In case of WGG37, more partitioning towards root components at delayed age of 69DAS and confinement of roots at the top soil depth due to sensitivity to water deficits at 22 DAS has led to reduced yields Table 1. Methodology Though every researcher appreciate the efficiency of 3D imaging, still 2D imaging techniques are easily accessible, technically simple Pierret et al. 2003 and may be considered as a possible relevant option for observing positioning of roots. In this methodology, although limitation in the size of root box is a constraint, care was taken to consider field level single plant spacing for the chamber size. 2D Root architecture image and root measurement at different soil profile depths could be possible in this methodology. The results suggest the suitability of the cultivar ML267 to conditions ensconced with intermittent moisture stress periods due to enhanced TRL, RLD and fine roots at deeper depths especially at 15-45cm and during water sufficient situations, WGG37 though performed significantly better than ML267 could further be improved by reducing more partitioning towards root at later stages of crop growth. Further, Root chamber-Pin board method may be helpful in studying the effect of soil and fertility factors on root architecture in addition to soil moisture. Therefore, this indigenized Root chamber-Pin board method is recommended for study of root architecture under controlled conditions. ACKNOWLEDGEMENT The authors are thankful to the SERC of ‘Department of Science and Technology, Govt. of India’ for funding the project on “Root Proliferation” and also to ICAR., India. REFERENCES Andersson A, Johansson E and Oscarson P. 2005. Nitrogen redistribution from roots in post-anthesis plants of spring wheat. Plant Soil 269: 321-332. Bauerle TL, Richards JH, Smart DR and Eissenstat DM. 2008. Importance of internal hydraulic redistribution for prolonging the lifespan of roots in dry soil. Plant,Cell Environment 31: 177–186. Campbell BD, Grime JP and Mackey JML. 1991. A trade-off between scale and precision in resource foraging. Oecologia 87: 532–538. Doussan C, Pagès L and Pierret A. 2003. 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Millsp. genotypes at early growth stages against soil moisture stress ANUJ KUMAR SINGH, J.P. SRIVASTAVA, R.M. SINGH 1 , M.N. SINGH 1 and MANOJ KUMAR 1 Department of Plant Physiology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi - 221005, Uttar Pradesh, India; Department of Genetics and Plant Breeding 1 , Institute of Agricultural Sciences, Banaras Hindu University, Varanasi - 221005, Uttar Pradesh, India; Email: anujkumarsinghbhu1gmail.com Received : July 11, 2013 ; Accepted : December 09, 2013 ABSTRACT Twenty pigeonpea genotypes were screened for terminal moisture deficit at early growth stages to identify suitable physiological parameters. Seeds were sown in plastic bags 30cm×15cm containing 2.5 kg soil. Moisture stress was imposed after 24 days of sowing. The physiological parameters viz ., relative water content RWC, cell membrane injury CMI due to thermal and osmotic stresses and chlorophyll stability index CSI were determined using first fully mature leaflets from top of the plants grown in net house. These parameters were correlated with their yield and yield attributes grown in field under rainfed conditions. Genotypes with higher RWC, lower CMI and high CSI during moisture deficit period exhibited higher pods plant -1 , seeds pod -1 and grain yield plant - 1 . These physiological traits may be utilized for development of high yielding drought tolerant varieties andor development of CMS based drought tolerant hybrids. Key words: CMI, CSI, RWC, correlation, moisture stress tolerance. Pigeonpea Cajanus cajan L. Millsp., is one of the major food legume crops of the tropics and sub-tropics. India has the largest acreage under pigeonpea 3.90 mha with a total production and productivity of 2.89 mt and 741 kg ha -1 , respectively DAC, 2011. Among several abiotic factors, drought is a key factor reducing crop productivity due to water loss which leads to decrease in CO 2 uptake adversely affecting rate of photosynthesis and stomatal conductance Federick et al. 1989. As pigeonpea is drought tolerant crop and has a large variation for maturity periods it is widely adapted to a range of environments and cropping systems. Broadly, four maturity groups are recognized in pigeonpea, i.e., extra early 90 – 120 days, early 120 – 150 days, medium 150 – 200 days and late 200 – 300 days. Variations of different maturity groups have direct relevance on the survival and fitness of the crop in different agro-ecological niches Choudhary, 2011. According to Kumar et al. 2011 a progressive water stress causes significant physiological and biochemical changes in pigeonpea. They suggested that RWC could be used to select high yielding genotypes under water deficit environment as they maintain high cell turgor. Cell damage due to abiotic stresses may also be quantified by measuring cell membrane injury and chlorophyll stability index Singh et al . 1992; Kumar et al. 2011. Chlorophyll stability during drought had been a promising criterion for selection against drought in peanuts Arunyanark et al. 2008. It had been pointed out that drought stress, imposed at different phonological stages influenced yield differently in pigeonpea Nam et al. 2001. In present study, an attempt was made to correlate the physiological parameters like cell membrane injury due to thermal and osmotic stress, chlorophyll stability index and relative water content under moisture stress at early phenological stages with yield and yield attributes in pigeonpea. MATERIALS AND METHODS Twenty pigeonpea genotypes viz., KPBR 80-2-1, SON 103, F 3 58-B, ICP 6579, IPA 16-F, ICP 11204, MA96 SBH-56, ICP 11887, NDA 1, MAL 13, BAHAR, MA 6, ICP 2506, MA98 PTH-2, SL 22-2-3, ICP 13857, JKM 7, ICP 5458, MA98 DEO-89 and ICP 8451 were selected for present study. The experiments were conducted in plastic bags 30cm×15cm, containing 2.5 kg soil in the wired net house as well as in field during 2011- 12 at Agricultural Research Farm, BHU, Varanasi and protected from rains using polythene sheets. After germination three healthy seedlings of uniform size were maintained in each bag. Moisture stress was created by withholding irrigation after 24 days of sowing. Observations were recorded at 6, 12 and 18 days after withholding irrigation pertaining to relative water content, cell membrane injury and chlorophyll stability index. All observations were recorded on first fully expanded leaflets from top of the plants in three replications. Leaf samples were collected in plastic bag between 10 – 11 AM. The leaf disks of 1 cm in diameter were used for the analysis. The relative water content RWC was estimated following procedure of Weatherley 1950 using the formula; RWC= Fresh weigh – Dry weigh Saturated weigh – Dry weigh × 100. The cell membrane injury due to osmotic or thermal stress was estimated in uppermost fully expanded leaf according to procedure of Blum and Ebercon 1981 and calculated by the formula CMI= [1-1-T 1 T 2 1-C 1 C 2 ] × 100, where, T and C refer to the conductivity of treatment and control and subscripts 1 and 2 indicates initial before autoclaving and final after autoclaving conductivity, respectively. The chlorophyll extraction was done by the 9 8 Journal of Food Legumes 263 4, 2013 method of Hiskox and Israelstam 1979 and total chlorophyll was calculated as per Arnon 1949. The chlorophyll destruction due to temperature stress was measured by method of Murthy and Majumdar 1962 called chlorophyll stability index CSI= C 1 C 2 × 100 where, C 1 and C 2 are chlorophyll contents in temperature treated and control samples, respectively. Above mentioned genotypes were also grown in Randomized Block Design under rainfed field condition with three replications and five plants were randomly selected from each genotype in each replication for yield and yield attributes viz., days to maturity, pods plant -1 , seeds pod -1 , 100-seed weight g and seed yield plant -1 g. RESULTS AND DISCUSSION Physiological parameters: Significant differences were recorded for RWC among genotypes, stages and their interactions Table 1. On an average RWC decreased steadily with an increase of stress duration. Through average performance over stages, it was observed that different genotypes had variable RWC. Some of the genotypes viz., KPBR 80-2-1, SON 103, F 3 58 B, ICP 6579, IPA 16-F, ICP 11204, NDA-1, MAL 13 and BAHAR maintained average RWC more than 80, while genotypes viz., ICP 13857, SL 22-2-3, ICP 5458 and ICP 8451 registered less than 70 and rest of the genotypes showed values between 70-80. It is interesting to note that KPBR 80-2-1 89.68 maintained the maximum RWC at all the stages, and ICP 8451 the minimum among all the studied genotypes. Decrease in relative water content, water potential, osmotic potential and turgor potential due to moisture stress has also been observed by other workers Coyne et al. 1982 and Kimani et al. 1994. If the ability to maintain high water status can be considered as an indication of drought tolerance, as suggested by Matin et al. 1989, then among the studied genotypes KPBR 80-2-1 may be ranked as the most tolerant and ICP 8451 as highly susceptible. Data regarding chlorophyll stability index CSI showed significant differences with respect to genotypes, stages and their interactions Table 2. Chlorophyll stability index decreased with the increase in stress duration. On an average CSI was the maximum in KPBR 80-2-1 91.20 and the minimum in ICP 8451 44.38. Genotypes KPBR 80-2-1, SON 103, F 3 58-B and ICP 6579 having more than 80 CSI after 18 days of stress show high stability of chlorophyll under thermal stress, while those with less than 50 ICP 13857, MA98-DE- O89 and ICP 8451 indicate susceptibility to thermal stress. Reduction in chlorophyll stability also indicates reduction of chlorophyll content as has been reported in drought stressed cotton Mssacci, 2008 Chlorophyll content also decreased significantly under higher water deficit in sunflower plants Kiani et al. 2008 and Vaccinium myrtillus Tahkokorpi et al. 2007. Table 1. Relative water content in leaves of pigeonpea genotypes at increasing moisture stress period. Stage Days after withholding irrigation Genotype 6 12 18 Mean KPBR80-2-1 95.50 77.75 91.23 72.74 86.00 68.03 86.00 68.03 89.68 SON103 94.00 75.82 90.23 71.76 81.50 64.53 80.34 63.65 86.52 F3 58B 85.54 67.62 81.34 64.38 85.54 67.62 80.56 63.79 83.25 ICP6579 91.00 72.54 79.56 63.08 80.50 63.79 77.56 61.68 82.16 IPA16F 90.56 72.05 83.56 66.03 80.20 63.58 76.45 60.94 82.69 ICP11204 85.65 67.80 85.64 67.70 70.76 58.00 79.50 45.00 80.39 MA96SBH56 85.00 67.21 84.00 66.42 71.35 57.61 70.00 56.79 77.59 ICP11887 89.56 71.09 75.50 60.33 81.23 64.30 71.20 57.54 79.37 NDA1 85.66 67.70 81.23 64.30 80.67 63.87 75.00 60.00 80.64 MAL13 91.00 72.54 85.00 67.21 74.50 59.67 70.10 56.85 80.15 BAHAR 88.89 70.45 82.00 64.90 86.45 68.44 77.00 61.34 83.59 MA6 88.50 70.18 71.00 57.42 75.50 60.33 55.00 47.87 72.50 ICP2506 91.45 72.95 77.00 61.34 75.00 60.00 70.00 56.79 78.36 MA98PTH2 81.34 64.45 72.34 58.31 65.54 54.03 65.00 53.73 71.06 SL22-2-3 85.00 67.21 73.00 58.69 66.00 54.33 53.00 46.72 69.25 ICP13857 88.86 70.45 70.00 56.79 64.80 53.61 55.65 48.22 69.83 JKM7 88.00 69.73 75.50 60.33 60.00 50.77 59.00 50.18 70.63 ICP5458 85.00 67.21 74.00 59.34 65.50 54.03 50.50 45.29 68.75 MA98DEO89 85.45 67.54 78.00 62.03 63.40 52.77 55.00 47.87 70.46 ICP8451 81.00 64.16 70.20 56.91 33.90 35.60 26.20 30.80 52.83 Mean 87.85 79.02 72.42 66.65 Particular SE d CD 1 Genotype 1.05 2.10 Stage 0.47 1.21 Genotype × Stage 2.10 5.40 The value in parenthesis represents the angular transformed values. Plants were grown in plastic bags of size 30cm×15cm containing 2.5 kg soil. Water deficit stress was imposed by withholding irrigation after 24 days of sowing. Singh et. al., : Selection parameters for pigeonpea Cajanus cajan L. Millsp. genotypes at early growth stages against soil 9 9 Table 2. Chlorophyll stability index of pigeonpea genotypes at incresing moisture stress period. Values in parentheses represent the angular transformed values. Plants were grown in plastic bags of size 30cm×15cm containing 2.5 kg soil. Water deficit stress was imposed by withholding irrigation after 24 days of sowing. Stage Days after withholding irrigation Genotype 6 12 18 Mean KPBR80-2-1 95.80 78.17 95.00 77.08 88.00 69.73 86.00 68.03 91.20 SON103 94.00 75.82 89.40 71.00 85.00 67.12 80.00 63.43 87.10 F3 58B 94.50 76.44 88.46 70.09 80.50 63.79 78.65 62.44 85.53 ICP6579 88.43 70.09 86.26 68.19 82.45 65.20 75.23 60.13 83.09 IPA16F 84.25 66.58 80.00 63.43 75.00 60.00 70.20 56.91 77.37 ICP11204 85.00 67.21 81.23 64.30 75.34 60.20 71.20 57.54 78.19 MA96SBH56 83.70 66.19 80.65 63.87 70.00 56.79 59.50 50.48 73.46 ICP11887 82.90 65.57 75.60 60.40 69.60 56.54 65.57 54.03 73.42 NDA1 88.24 69.91 78.00 62.03 68.76 55.92 72.45 58.31 76.86 MAL13 81.00 64.16 70.60 57.17 65.50 54.03 60.40 51.00 69.38 BAHAR 81.80 64.75 71.50 57.73 60.90 51.30 51.00 45.57 66.30 MA6 79.80 63.29 67.80 55.43 55.00 47.87 48.50 44.14 62.78 ICP2506 80.14 63.43 65.40 53.97 50.45 45.43 45.60 42.48 60.40 MA98PTH2 79.50 63.08 62.40 52.18 45.50 42.42 39.00 38.65 56.60 SL22-2-3 77.70 61.80 58.30 49.80 40.80 39.70 33.70 35.50 52.63 ICP13857 75.50 60.33 55.60 48.22 35.00 36.27 29.80 33.09 48.98 JKM7 85.50 67.62 60.90 51.30 30.65 33.58 30.90 33.71 51.99 ICP5458 84.40 66.66 58.90 50.13 35.67 36.63 25.80 30.53 51.19 MA98DEO89 85.10 67.29 65.00 53.73 25.10 30.65 18.80 25.76 48.50 ICP8451 86.00 68.03 61.80 51.83 19.20 26.00 10.50 18.90 44.38 Mean 84.66 72.64 57.92 52.64 Particular SE d CD 1 Genotype 0.76 1.96 Stage 0.34 0.88 Genotype × Stage 1.53 3.93 The value in parenthesis represents the angular transformed values. Plants were grown in plastic bags of size 30cm×15cm containing 2.5 kg soil. Water deficit stress was imposed by withholding irrigation after 24 days of sowing. Table 3. Cell membrane injury due to osmotic stress in leaf tissues of pigeonpea genotypes at increasing moisture stress period. Stage Days after withholding irrigation Genotype 6 12 18 Mean KPBR80-2-1 7.86 16.22 11.80 20.09 15.60 23.26 20.90 27.20 14.04 SON103 15.00 22.79 22.00 27.97 25.00 30.00 31.0033.83 23.25 F3 58B 17.80 24.95 21.20 27.42 28.50 32.27 28.00 31.95 23.88 ICP6579 16.88 24.20 25.50 30.33 35.00 36.27 38.00 38.06 28.85 IPA16F 15.90 23.50 28.45 32.20 34.50 35.97 37.00 37.46 28.96 ICP11204 17.80 24.95 33.00 35.06 29.00 32.58 42.00 40.40 30.45 MA96SBH56 19.87 26.42 24.50 29.67 44.00 43.85 46.0042.71 33.59 ICP11887 17.80 24.95 28.00 31.95 45.30 42.30 48.50 44.14 34.90 NDA1 18.45 25.40 29.00 32.58 42.00 40.40 50.00 45.00 34.86 MAL13 19.50 26.21 35.00 36.27 49.50 44.71 51.50 45.86 38.88 BAHAR 21.00 27.27 35.50 36.57 45.50 42.42 55.50 48.16 39.38 MA6 19.20 25.99 38.00 38.06 45.00 42.13 50.50 45.29 38.18 ICP2506 20.00 26.57 35.27 36.39 48.51 44.14 50.00 45.00 38.45 MA98PTH2 18.70 25.62 35.00 36.27 54.00 47.29 58.56 49.89 41.57 SL22-2-3 14.56 22.38 36.00 36.87 58.67 49.95 61.55 51.65 42.70 ICP13857 18.97 25.77 37.80 37.94 49.00 44.43 56.80 50.07 40.64 JKM7 17.80 24.95 36.50 37.17 51.00 45.57 54.00 47.29 39.83 ICP5458 22.34 28.18 37.98 38.00 54.00 47.29 55.00 47.87 42.33 MA98DEO89 21.80 27.83 41.89 40.28 55.50 48.16 60.00 50.77 44.80 ICP8451 23.00 28.66 42.34 40.57 65.00 53.73 72.00 58.05 50.59 Mean 17.34 30.51 42.22 46.90 Particular SE d CD 1 Genotype 0.59 1.51 Stage 0.26 0.67 Genotype × Stage 1.17 3.01 100 Journal of Food Legumes 263 4, 2013 Table 4 . Cell membrane injury due to thermal stress in leaf tissues of pigeonpea genotypes at increasing moisture stress period Stage Days after withholding irrigation Genotype 6 12 18 Mean KPBR80-2-1 9.00 17.46 11.50 19.80 24.80 29.87 32.57 34.76 19.47 SON103 13.50 21.72 24.70 29.36 42.54 40.69 57.80 49.49 34.64 F358B 14.50 22.38 26.80 31.20 33.76 41.38 35.90 36.80 30.24 ICP6579 17.80 24.95 28.43 32.20 42.89 40.86 49.50 44.71 34.66 IPA16F 18.50 25.47 32.67 34.82 41.40 40.05 48.00 43.85 35.14 ICP11204 18.00 25.10 32.00 34.45 42.80 40.86 47.80 43.74 35.15 MA96SBH56 17.89 24.95 35.00 36.27 46.00 42.71 51.25 45.69 37.54 ICP11887 19.50 26.21 40.00 39.23 48.56 44.14 55.50 48.16 40.89 NDA1 19.90 26.49 36.00 36.87 45.67 42.48 58.00 49.60 39.89 MAL13 23.40 28.93 37.80 37.94 48.70 44.26 55.00 47.87 41.23 BAHAR 26.00 30.66 38.50 38.35 48.00 43.85 52.50 46.43 41.25 MA6 22.60 28.39 42.80 40.86 51.50 45.86 58.00 49.60 43.73 ICP2506 27.50 31.63 41.20 39.93 50.34 45.17 56.56 48.73 43.90 MA98PTH2 26.50 30.98 43.76 41.38 50.67 45.34 65.34 53.91 46.57 SL22-2-3 27.80 31.82 44.50 41.84 58.00 49.60 68.00 55.55 49.58 ICP13857 25.30 30.20 45.80 42.59 59.00 50.18 65.00 53.73 48.78 JKM7 19.90 26.49 44.67 41.90 61.00 51.35 68.00 55.55 48.39 ICP5458 28.70 32.39 49.76 44.83 64.50 53.43 68.50 55.86 52.87 MA98DEO89 26.80 31.18 47.65 43.62 64.89 53.81 80.60 63.90 54.99 ICP8451 25.00 30.00 48.90 44.37 65.76 54.15 84.40 66.70 56.02 Mean 20.39 36.12 48.23 56.01 40.18 Particular SE d CD 1 Genotype 0.38 0.99 Stage 0.17 0.44 Genotype × Stage 0.77 1.98 The value in parenthesis represents the angular transformed values. Plants were grown in plastic bags of size 30cm×15cm containing 2.5 kg soil. Water deficit stress was imposed by withholding irrigation after 24 days of sowing. Table 5. Yield and yield attributes of pigeonpea genotypes under rainfed condition. Genotype Days to maturity Pods plant -1 Seeds pod -1 Test weight g Seed yield plant -1 g KPBR 80-2-1 234.33 802.33 3.60 12.47 173.29 SON 103 244.67 386.67 4.00 12.58 165.56 F3 58 B 225.33 726.17 3.37 9.37 161.44 ICP 6579 236.00 786.00 3.57 7.62 147.05 IPA 16F 229.00 577.56 3.59 9.69 143.88 ICP 11204 235.33 728.33 3.70 8.47 137.21 MA96SBH56 233.67 411.00 3.63 10.65 117.27 ICP 11887 212.00 601.33 3.23 7.88 93.82 NDA 1 226.44 211.11 3.74 10.06 77.73 MAL 13 205.44 266.76 3.41 13.73 67.99 BAHAR 242.00 378.30 3.37 11.51 66.26 MA6 225.33 283.89 3.27 10.73 56.93 ICP 2506 213.00 365.17 3.30 7.21 60.50 MA 98PTH2 224.67 112.67 4.52 15.57 52.16 SL 22-2-3 243.00 140.33 4.32 12.28 47.78 ICP 13857 239.67 372.33 4.26 7.21 31.06 JKM 7 230.00 105.33 5.76 13.35 33.40 ICP 5458 211.67 155.33 5.21 10.96 28.70 MA 98DEO89 225.00 104.93 4.32 9.72 17.66 ICP 8451 222.00 152.33 3.57 7.41 15.76 SE mean± 0.090 53.35 0.17 0.10 1.12 CD 5 0.183 108.04 0.35 0.21 2.26 Plants were grown in field under rainfed condition with three replications and five plants from each genotype were selected in each replication for yield and yield attributes. Singh et. al., : Selection parameters for pigeonpea Cajanus cajan L. Millsp. genotypes at early growth stages against soil 101 Per cent cell membrane injury CMI due to osmotic stress in first fully expanded leaves from top is presented in Table 3. Among genotypes, stages and their interactions the significant differences were observed for this trait. It is observed that CMI due to osmotic stress intensified with advancement in stress duration. On an average, the genotypes viz ., KPBR 80-2-1, SON 103, F 3 58-B, ICP-6579, IPA 16-F, ICP 11204, ICP 11887, MA96 SBH-56 and NDA 1 exhibited less than 35 CMI due to osmotic stress, whereas, genotypes MAL 13, BAHAR, MA 6 and ICP 2506, ICP 13857, SL 22-2-3, JKM 7, ICP 5458, MA 98-PTH-2, MA 98-DEO-89 and ICP 8451 exhibited 40 or more CMI due to osmotic stress. Saadalla et al . 1990 reported that heat tolerant genotypes of wheat on the basis of electrolyte leakage, out yielded sensitive ones by 19 under field conditions. Kuo et al. 1993 showed that vegetable species with low cell membrane injury were more stable in different growing seasons. Significant differences were recorded in CMI with respect to genotypes, stages and their interactions due to increase in thermal stress duration Table 4. On an average, CMI due to thermal stress was minimum 19.47 in genotype KPBR 80-2-1 and the maximum in genotype ICP 8451 56.02. The genotypes KPBR 80-2-1, SON 103, F 3 58-B, ICP 6579, IPA 16-F, ICP 11204, MA96 SBH-56 and NDA 1 registered less than 40 CMI whereas, ICP 5458, MA98 DEO-89 and ICP 8451 showed more than 50 CMI due to thermal stress. CMI analysis being simple, rapid and reproducible is ideal for screening large populations. The results suggest that the temperatures in the range of 45 to 55 °C are suitable for determining genotypic variation. It is interesting to note that genotypes which had higher chlorophyll stability index CSI also had less cell membrane injury CMI due to thermal as well as osmotic stress and vice-versa. Yield attributes and correlation with physiological traits: Significant genotypic differences were observed for 20 genotypes under rainfed condition in respect of various traits Table 5. The maturity duration of genotypes ranged from 205 MAL 13 to 244 SON 103 days. Pods plant -1 was the minimum in genotype MA98DEO-89 104 and the maximum in KPBR 80-2-1 802. It was evident that genotypes with relatively longer duration generally produced more pods. The maximum seeds pod -1 were recorded in genotype JKM 7 5.76 and the minimum in ICP 11887 3.23. 100-seed weight was maximum in genotype MA98 PTH-2 15.57 g and the minimum in ICP 2506 7.21 g. Seed yield plant -1 ranged from 15.76 g to 173.29 g, being the maximum in KPBR 80-2-1 173.29 g and the minimum in ICP 8451 15.76 g. The genotypes KPBR 80-2-1, SON 103, F 3 58-B and ICP 6579 having high grain yield plant -1 also had high 100-seed weight, seeds pod -1 and pods plant -1 and high RWC, low CMI due to osmotic as well as thermal stresses and high chlorophyll stability during increasing moisture deficit. Reddy 2001 reported genotypes LRG 30, ICPL 85063 and ICPL 332 as the most suitable for cultivation under rainfed condition, as they maintained higher RWC and yielded more under soil water deficit. On the basis of present investigation, it is opined that higher RWC in water deficit tolerant genotypes leads to maintenance of higher cell membrane stability and chlorophyll integrity, resulting in higher pods plant -1 and thereby higher yield. It is also observed that physiological traits viz., RWC, CSI are positively and significantly associated with pods plant -1 and seed yield plant - 1 and CMI due to osmotic and thermal stress is negatively correlated with pods plant -1 and seed yield plant -1 Table 6. In general, high RWC and CSI along with low CMI due to osmotic and thermal stress were exhibited by genotypes SON 103, KPBR 80-2-1, F 3 58-B, ICP 6579, IPA 16-F and ICP 11204. These genotypes belong to medium to late duration groups and gave higher seed yield plant -1 ranging from 137.21 to 173.29 g, under rainfed condition. Thus, these genotypes may be used for development of high yielding drought tolerant variety andor development of CMS based drought tolerant hybrids. Physiological traits like RWC, CSI and CMI due to thermal or osmotic stresses may be used for identification of suitable genotypes for rainfed conditions. ACKNOWLEDGEMENT Authors greatly acknowledge the financial support from U.P. Council of Agricultural Research UPCAR, Lucknow under Sodh Nidhi Project ‘Development of Drought Tolerant Cultivars of Pigeonpea’ and the Banaras Hindu University for providing infrastructure facilities. REFERENCES Arnon DI. 1949. 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Significant correlation Parameter Days to maturity Pods plant -1 Seeds pod -1 Test weight Seed yield plant -1 RWC 0.21 0.69 -0.40 0.13 0.81 CMI due to Osmotic Stress -0.30 -0.81 0.32 -0.06 -0.94 CMI due to Thermal Stress -0.26 -0.84 0.47 -0.07 -0.94 CSI 0.21 0.80 -0.51 0.02 0.96 102 Journal of Food Legumes 263 4, 2013 DAC 2011. Fourth Advance Estimates of Production of Food grains for 2010-11. Agricultural Statistics Division, Directorate of Economics Statistics, Department of Agriculture Cooperation, Government of India, New Delhi. Federick JR, Alm DM and Hesketh, JD. 1989. Leaf photosynthetic rates, stomatal resistance and internal CO 2 concentrations of soybean cultivars under drought stress. Photosynthetica 23: 575-584. Hiskox JD and Israelstam GF. 1979. A method for the extraction of chlorophyll from leaf tissue without maceration. Canadian Journal of Botany 57: 1332-1334. Kiani SP, Maury P, Sarrafi A and Grieu P. 2008. 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Cell membrane stability in relation to drought tolerance in wheat genotypes. Journal of Agronomy and Crop Science 168: 186-190. Tahkokorpi M, Taulavuori K, Laine K and Taulavuori E. 2007. After- effects of drought related winter stress in previous and current year stems of Vaccinium myrtillus L. Environmental and Experimental Botany 61: 85–93. Weatherley PE. 1950. Studies in the water relations of the cotton plant. I. The field measurement of water deficits in leaves. New Phytologist 49: 81-87. Journal of Food Legumes 263 4: 103-114, 2013 Optimization of extrusion process variables for development of pulse-carrot pomace incorporated rice based snacks MD. SHAFIQ ALAM , BALJIT SINGH 1 , HARJOT KHAIRA, JASMEEN KAUR and SUNIL KUMAR SINGH Department of Processing and Food Engineering, Punjab Agricultural University, Punjab, India; 1 Department of Food Science and Technology, Punjab Agricultural University, Punjab, India; E-mail: ms_alamrediffmail.com Received : April 10, 2013 ; Accepted : August 09, 2013 ABSTRACT Different experimental combinations of extrusion process variables i.e. screw speed, die temperature, moisture content and proportion of ingredients were tried using Box- Behnken design of experiments. Response surface methodology RSM was used to investigate the effect of screw speed 300-500 rpm, die temperature 120-180° C, moisture content 14-20 and proportion of ingredients rice flour: pulse flour: carrot pomace powder- 60-80: 10-30: 10 on protein, fiber, overall acceptability, colour and texture of extruded products. The extrusion process was optimized for maximum protein, fiber, overall acceptability and minimum hardness with colour difference within experimental range. The optimum operating conditions using selective quality parameters for screw speed, die temperature, moisture content and rice proportion in ingredient composition was 340 rpm, 120°C, 20 and 60, respectively. An analysis of variance ANOVA revealed that among the process variables, sample formulation followed by screw speed had the most significant effect on all the responses; die temperature and moisture content had the significantly higher effect on overall acceptability and hardness. Keywords: Carrot pomace, Extrusion, Overall acceptability, Pulse flour, Texture Extrusion cooking technology is a versatile and efficient method of converting raw materials into finished food products. It can replace many conventional processes in food and feed industry because of its uniqueness among the heat processes by subjecting the moistened starchy or proteinaceous foods to intense mechanical shear resulting into viscous, plastic-like dough which cooks before being forced through the die. Extrusion cooking has been widely used in formation of various types of products. However, the incorporation of fruit and vegetable wastes and pulses in rice based extruded products to make a healthy nutritious snack is still not fully explored. The food processing industry produces large quantities of waste by-products. They are inexpensive, available in large quantities, characterized by a high dietary fiber content resulting with high water binding capacity and relatively low enzyme digestible organic matter Serena and Bach-Knudsen 2007. Due to the high dietary fiber content and contrasting dietary fiber properties, the co-products could be used to change physicochemical properties of diets. A number of researchers have used fruits and vegetable by-products such as apple, pear, orange, peach, blackcurrant, cherry, artichoke, asparagus, onion, carrot pomace Nawirska and Kwasnievska 2005, Grigelmo-Miguel and Martin-Belloso 1999, Ng et al. 1999 as sources of dietary fiber supplements in food. Carrot pomace is a by-product obtained during carrot juice processing. The juice yield in carrots is only 60–70, and even up to 80 of carotene may be lost with left over carrot pomace Bohm et al. 1999. The carrot pomace has good residual amount of all the vitamins, minerals and dietary fiber. The dried carrot pomace can be used to develop high in fiber content extruded products. The extruded products which are high in fiber content can be further improved in its overall nutritional quality and taste by the incorporation of pulses. The pulses in the extruded product can pave the way for a snack which is rich in both fiber and protein content. Lentil also known as red dahl, masur, massar, and split pea high in protein especially rich in lysine and leucine, low in fat, and is an excellent source of dietary ûber and complex carbohydrates. Lentil also contains vitamins and minerals such as B vitamins, calcium, phosphorous and potassium, along with oleic, linoleic and palmitic acid Adsule 1996, Agriculture and Agri-Food Canada 2006. The nutritional value of lentil and its use in a variety of culinary applications make it an important commodity in terms of production, and trade. Every extruded product needs a base raw material which provides the overall structure to the product. The major waste product of the cereal industry is broken rice kernels. The broken rice is a by-product of modern rice milling process. The rice portion can have varying percentages 5 - 7 of broken kernels which contain nutritive value similar to whole rice and are available readily at relatively lower cost. These can be easily used in the formation of rice flour which is an attractive ingredient in the extrusion industry due to its bland taste, attractive white color, hypoallergenicity and ease of digestion Kadan et al. 2003. Keeping these points in mind the present study was planned with an objective to develop a novel extruded product from pulse flour, carrot pomace and rice flour and to study the effect of extrusion process variables on quality attributes of developed snacks. 104 Journal of Food Legumes 263 4, 2013 MATERIALS AND METHODS Experimental design: Response surface methodology RSM was adopted in the design of experimental combinations Montgomery 2001, Ding et al. 2005, Altan et al. 2008 a and b, Yagci and Gogus 2008, Alam et al. 2011. A four-factor three levels Box- Behnken experimental design was employed Table 1. The ingredients used for the carrot pomace based ready- to-eat snack preparation were: Rice flour R; pulse powder P, red lentil and carrot pomace C. The independent variables included the ingredient composition pulse powder 10-30, rice flour 60-80 and carrot pomace 10, moisture content 14–20, screw speed 300-500 rpm and die temperature 120- 180°C. The three levels of the process variables were coded as -1, 0, +1 Montgomery 2001.The developed extruded product was evaluated on the basis of response variables like colour L, a, b, colour difference, chroma and hue angle, texture hardness, adhesiveness, springiness, cohesiveness, gumminess, chewiness and resilience and overall acceptability. Sample preparation: Ingredient formulations for conducting extrusion experiments are given in Table 2. Rice flour R was replaced with pulse powder P at levels of 10, 20 and 30. In each sample, carrot pomace C was added at the level of 10 in order to increase the fibre content. Composite flour 300 gm was prepared for each sample. In order to enhance the taste of developed product 2 salt was added to each sample. After mixing, samples were stored in polyethylene bags at refrigerated temperature for 24 h Stojceska et al. 2008. The moisture content of all the samples was determined after preparation by halogen moisture analyzer Make: Mettler Toledo, HR83 Halogen prior to extrusion experiments. Levels Independent Variables Symbol Coded Uncoded 1 500 400 Screw Speed rpm A -1 300 1 180 150 Die Temperature °C B -1 120 1 20 17 Moisture Content C -1 14 1 80:10:10 70:20:10 Proportion of Ingredients-R: P: C D -1 60:30:10 Table 1. Box-Behnken design for response surface methodology Ingredients preparation Dry carrot pomace powder preparation: Commercial variety of carrot was procured from local market, Ludhiana, India. These were washed in running tap water two times to remove extraneous material. Trashes were removed with a plane stainless steel knife and trimming was also done. A grinder Make: Sujata 750 W was used to extract carrot juice. The pomace was collected for further studies. The carrot pomace was pretreated with 1 wv citric acid. The pretreated pomace was then kept in a tray dryer at 65°C to bring the desired moisture content of dried carrot pomace to 6.0 d.b. The dried pomace was ground to powder using the same grinder Make: Sujata 750 W. The pomace powder was stored in sealed laminated aluminum films for further use. Rice and pulse flour preparation: The broken rice and pulse brokens were procured from local market in Ludhiana, Punjab. Those samples were then ground to powder with grinder Make: Sujata 750 W. Extrudates preparation Extrusion of samples was performed using laboratory scale co-rotating twin- screw extruder with intermeshing screws Model BC21; Clextral, Firminy Cedex, France. The length to diameter LD ratio of extruder was 16:1. Temperature of the first, second and third zone was maintained at 40, 70 and 100° C respectively throughout the experiments, while the temperature of last zone was varied according to experimental design. The diameter of die opening was 6mm.The extruder was thoroughly calibrated with respect to the combinations of feed rate and the screw speed to be used. A variable speed die face cutter with four blade knives was used to cut the extrudates. The product was collected at the die end and kept at 60 ± 0.5°C in an incubator for 1 h duration to remove extra moisture from the product. The samples were packed in polythene bags for further analysis. Quality Parameters Crude Protein: Macro-kjeldhal method was used to determine nitrogen. Conversion factor of 5.95 and 6.25 was used for crude protein estimation of extruded products. 1g of grounded sample was digested in Kjeldhal flask with digestion mixture copper sulphate and potassium sulphate in 1:9 ratio and concentrated H 2 SO 4 20 ml till light green colour appeared and finally cooled. Ammonia released by distillation of digested samples with saturated NaOH 80 ml was captured in 0.1 N HCl and percent N was estimated. The protein content was calculated as per cent nitrogen × factor Ranganna 1997. Crude Fiber: Crude fibre of extruded snacks was estimated using Fibertec Foss instrument, Sweden. Capsules for holding the sample were kept in hot air oven at 100 °C for 20 minutes for drying, cooled and weighed. One gram of the grounded sample was weighed in the capsule Defatting of Table 2. Ingredient formulations Proportion of ingredients Blends- R:P:C Ingredients 60:30:10

70:20:10 80:10:10

Rice gm 180 210 240 Red Lentil gm 90 60 30 Carrot Pomace gm 30 30 30 Total gm 300 300 300 Alam et. al. : Optimization of extrusion process variables for development of pulse-carrot pomace incorporated rice based 105 samples was done if necessary. Capsules were fixed in the rotating stand and put it into the extraction cup; 250 - 275 ml of 1.25 per cent H 2 SO 4 was added to the extraction cup and immersed the stand into the beaker. Acid extraction was done by boiling it for 30-40 minutes followed by its washing with hot water. Then alkali washing was done with 1.25 per cent NaOH for the same time duration followed by hot water washing. Finally, capsules were dried in oven for 2 hours at 130 °C and then placed at 550 °C for 5 hours, cooled and weighed for crude fibre estimation AOAC 2005. Texture Profile Analysis TPA Textural attributes of extrudates were determined by texture profile analysis TPA using Texture Analyzer, model TA-XT2i Stable Micro-Systems, Surrey, England equipped with a compression plate P75. The tests were conducted at pre–test speed of 1.0 mmsec, test speed of 5 mmsec, post test speed of 5 mms at strain of 25 and trigger force of 0.4903 N using load cell of 50kg. Hardness: Mechanical properties of the extrudates were determined by crushing method using a TA-XT2i Stable Micro-Systems, Surrey, England with a compression plate of 75 mm diameter Kumar et al. 2009. The tests were conducted at pre–test speed of 1.0 mmsec, test speed of 5 mmsec, post test speed of 5 mms, strain- 25, trigger force of 0.4903 N and load cell of 50 kg. The highest first peak value was recorded as this value indicated the first rupture of snack at one point and this value of force was taken as a measurement for hardness Stojceska et al. 2008 Cohesiveness: The ratio of positive force areas under the second and first compression A2A1 was defined as cohesiveness. Firmness: The height of the force peak on the first compression cycle first bite was defined as firmness N. Springiness or elasticity: The distance that the sample recovered its height during the time elapsed between the end of the first bite ad start of the second bite mm. Gumminess: It is defined as the product of firmness x cohesiveness N. Chewiness: It is defined as the product of hardness x cohesiveness x springiness N-mm Adhesiveness: The work necessary to overcome the attractive forces between the surface of the food and the surface of other materials with which the food comes into contact e.g. tongue, teeth, palate. Colour: The colour property of extruded samples was measured by using Colour Reader CR-10 Konica Minolta Sensing Inc. Japan. For determination of colour, the sample was ground to powder with the help of Grinder Make: Sujata 750 W. The powder was completely filled in petridish provided that no light is allowed to pass during the measuring process. The ‘L’, ‘a’ and ‘b’ values were recorded at D 6510°. The colour difference was measured by the equation given by Gnanasekharan et al. 1992. Colour difference = [ L-L 2 + a-a 2 + b-b 2 ] OR Colour difference = [ L-L 2 + a-a 2 + b-b 2 ] 12 Where; L , a and b represent the respective readings of developed raw sample before extrusion. The chroma and Hue angle were estimated as- Chroma = a 2 + b 2 Chroma = [ a2 + b2] 12 Sensory evaluation: Hedonic scale evaluation method for sensory analysis was used to find out the order of preference of extruded samples. The samples were evaluated for sensory quality on the basis of overall acceptability on a 9 point hedonic scale 9-liked extremely to 1-disliked extremely on the basis of colour, flavour and mouthfeel from semi-trained penal of 10 panelists according to the method described by Amerine et al. 1965. Optimization of process parameters: Response surface methodology was applied to the experimental data using a commercial statistical package, Design-Expert version 8.0.7.1 Statease Inc, Minneapolis, USA, Trial version. The same software was used for the generation of response surface plots, superimposition of contour plots and optimization of process variables. The response surface and contour plots were generated for different interaction for any two independent variables, while holding the value of other two variables as constant at the central value. Such three- dimensional surfaces could give accurate geometrical representation and provide useful information about the behavior of the system within the experimental design Cochran and Cox et al. 1964. The optimization of the extrusion process aimed at finding the levels of independent variables viz. Moisture content, Screw speed, Die temperature, and rice flour, which could give maximum protein, fiber and overall acceptability; and minimum hardness with colour change in range. Desirability, a mathematical method was used for selecting the optimum process values. For several responses and factors, all goals get combined into one desirability function. The numerical optimization finds a point that maximizes the desirability function. RESULTS AND DISCUSSION Protein Content: The protein content of extruded snacks ranged from 11.74 to 7.89 table 3. The results revealed that the ingredient composition and screw speed had a significant impact on the protein content. The other extrusion parameters viz. die temperature and moisture content had a non-significant impact on protein content of extrudates table 2. 106 Journal of Food Legumes 263 4, 2013 Contour plots reveal that protein content decreased with the increase in rice percentage in ingredient composition fig 1. For a given value of screw speed i.e. 300 rpm, the protein decreased from 11.56 for ingredient proportion containing 60 rice to 10.32 for ingredient proportion containing 70 rice and further to 8.26 for ingredient proportion containing 80 rice. This trend can be easily explained by the decrease in the percentage of pulse flour. The screw speed also had a significant impact on the protein content of extrudates. The protein content in extrudates decreased consistently with the increase in screw speed. Contour plots reveal that for given moisture content of 14, the protein content decreased from 10.25 at the screw speed of 300 rpm to 9.93 at the screw speed of 400 rpm. This decrease further continued to 9.54 at 500 rpm screw speed. Fiber content: The fiber content of the snacks ranged between 11.92 to 9.89 respectively with significant impact of ingredient proportion and screw speed table 3. The other Fig 1: Contour plots depicting the effect of process parameters on protein content processing parameters viz. die temperature and moisture content had a non-significant impact on the fiber content of the extrudates table 2. Contour plots reveal that fiber content decreased with the increase in rice percentage in ingredient composition fig 2. For a given value of screw speed i.e. 300 rpm, the protein decreased from 12.00 for ingredient proportion containing 60 rice to 10.78 for ingredient proportion containing 70 rice and further to 10.17 for ingredient proportion containing 80 rice. The screw speed also had a significant impact on the protein content of extrudates. The protein content in extrudates decreased consistently with the increase in screw speed. Contour plots reveal that for given moisture content of 14, Fig 2. Contour plots depicting the effect of process parameters on fiber content Alam et. al. : Optimization of extrusion process variables for development of pulse-carrot pomace incorporated rice based 107 the protein content decreased from 10.46 at the screw speed of 300 rpm to 10.26 at the screw speed of 400 rpm. This decrease further continued to 9.54 at 500 rpm screw speed. Overall acceptability: The overall acceptability for extruded samples ranged from 4.33 to 8.33 on a 9 point hedonic scale Table 4. The process parameters viz. moisture content, screw speed and die temperature had a significant P0.05 effect on overall acceptability of extruded samples whereas the effect due to ingredient composition was non- significant Table 3. Contour plots reveal that overall acceptability of the product increased with increase in screw speed fig 3. For a given moisture content of 14, the overall acceptability increased from 5.78 at 300 rpm to 7.10 at 400 rpm and finally to 8.58 at 500 rpm respectively. This may be explained by better mixing of components during intensive baro-thermal treatment higher shearing stress, formation of starch-protein matrix during processing at the temperature higher than starch gelatinization temperature and higher moisture content in raw materials Moscicki et al. 2007. On the other and the overall acceptability first decreased and then increased with the subsequent increase in moisture content. For a given value of screw speed i.e. 300 rpm, the overall acceptability was 5.78 at 14.5 moisture content which decreased to 5.09 at 17 moisture content. With further increase in the moisture content the overall acceptability indicated a slight increase to 5.61 at 20 moisture content. Contour plots reveal that overall acceptability increased with the increase in the die temperature. At a given ingredient proportion containing 60 rice the overall acceptability increased from 5.66 to 5.98 with increase in die temperature from 120 to 150°C. The overall acceptability further increased to 6.81 with the increase in die temperature to 180°C. Although the effect of ingredient composition on overall acceptability was non-significant but it was observed that the overall acceptability decreased with the increase in percentage of Note: , significant at P0.05 and P0.01 respectively; Least Squares Means analysis. Protein Fiber Overall Acceptability Extrusion Process Variables F value F value F value Moisture Content A 1.63 2.54 14.24 Screw Speed B 90.13 101.96 42.67 Die Temperature C 0.00 0.00 23.76 Ingredient Composition-R:P:C D 1453.55 739.69 7.559 AB 0.02 0.02 3.38 AC 4.40 0.13 0.69 AD 0.41 0.22 0.029 BC 1.53 0.10 0.54 BD 7.27 11.45 0.69 CD 0.20 0.46 0.24 A 2 2.72 2.83 8.08 B 2 0.28 3.14 0.15 C 2 0.07 3.36 1.35 D 2 47.99 52.89 0.020 C.V 1.37 0.90 7.81 S.D p=0.05 0.13 0.096 0.50 Table 3. Analysis of variance of extrusion process variables on protein, fiber and overall acceptability Fig 3. Contour plots depicting the effect of process parameters on overall acceptability 108 Journal of Food Legumes 263 4, 2013 rice in ingredient composition. For a given value of die temperature i.e. 120°C, the overall acceptability decreased from 5.66 at 60 rice proportion in ingredient composition to 5.39 at 80 rice proportion in ingredient composition. Colour attributes of extrudates The colour difference “E in extruded samples when compared to the fresh samples ranged from 7.6 to 16.55 Table 6. The overall effect of moisture content, die temperature screw speed and flour composition on colour difference was non-significant Table 5. The colour difference first increased and then decreased with the increase in moisture content fig 4. For a given value of screw speed i.e. 300 rpm, the colour difference increased from 10.53 at 14 moisture content to 12.39 at 17 moisture content. The colour difference further decreased to 10.49 at 20 moisture content. On the other hand colour difference increased with the subsequent increase in screw speed. For the given value of moisture content 14, the colour difference increased from 10.53 to 11.64 for the increase in screw speed from 300 to 500 rpm. Table 4. Experimental data for protein, fiber and overall acceptability using four factor three level Box- Behnken design Extrusion Process Variables Moisture content Screw speed rpm Die temp °C Ingredient Composition- R:P:C Protein Fiber Overall Acceptability 17 400 150 70:20:10 10.01 10.47 5.5 14 400 120 70:20:10 9.83 10.51 7.5 17 300 150 60:30:10 11.74 11.92 5.33 17 400 150 70:20:10 9.99 10.4 6.17 17 300 120 70:20:10 10.24 10.83 4.33 14 400 150 60:30:10 11.02 11.56 6.5 17 400 150 70:20:10 9.94 10.44 6.17 20 300 150 70:20:10 10.2 11.01 6 17 500 150 60:30:10 10.62 11.02 7.17 17 400 120 80:10:10 8.11 10.16 5.17 17 300 150 80:10:10 8.29 10.21 5 20 400 180 70:20:10 9.8 10.62 7 20 400 150 60:30:10 10.74 11.6 5.83 20 400 120 70:20:10 10.11 10.57 5.5 17 300 180 70:20:10 10.12 10.76 5.5 14 400 150 80:10:10 8.09 9.89 6.67 17 400 120 60:30:10 10.93 11.61 5.83 17 500 180 70:20:10 9.74 10.29 8 17 500 120 70:20:10 9.53 10.3 6.1 20 400 150 80:10:10 7.98 10.02 6.17 17 400 180 80:10:10 8.02 10.11 7 14 400 180 70:20:10 10.08 10.49 8.17 17 500 150 80:10:10 7.89 9.96 7.67 20 500 150 70:20:10 9.29 10.39 6.33 14 500 150 70:20:10 9.41 10.32 8.33 17 400 180 60:30:10 10.96 11.69 7.17 14 300 150 70:20:10 10.28 10.91 6.17 Fig 4. Contour plots depicting the effect of process parameters on colour difference Alam et. al. : Optimization of extrusion process variables for development of pulse-carrot pomace incorporated rice based 109 The colour difference decreased and then increased with the increase in die temperature. According to contour plots, at a given value of rice percentage in ingredient proportion i.e. 60, the colour difference decreased from 15.72 at 120°C to 13.69 at 150°C. This value indicated a slight increased to 13.85 till the die temperature of 180°C. The results for L-value for extruded samples ranged from 70.95 to 78.1 with most of the samples lying in the range of 74 to 76 Table 6. There was an almost non-significant effect of various levels of screw speed, moisture contents and die temperatures on the L-value of extrudates Table 5. The highest L-values above 76 were attained above screw Table 5. Analysis of variance of extrusion process variables on colour attributes Colour Attributes L a b ? E Chroma Hue Angle Extrusion Process Variables F value F value F value F value F value F value Moisture Content A 0.45 6.72 12.09 0.45 11.92 4.83 Screw Speed B 3.02 0.45 0.033 0.034 0.052 0.85 Die Temperature C 0.08 1.72 1.53 0.051 1.64 1.27 Ingredient Composition-R:P:C D 4.991 2.90 8.77 3.41 8.35 1.20 AB 1.497 1.48 0.70 0.14 0.75 2.39 AC 0.066 1.48 0.050 0.41 0.11 1.91 AD 0.23 0.073 0.31 0.25 0.29 0.057 BC 0.18 0.036 0.87 0.14 0.75 0.013 BD 5.989 0.018 0.089 0.14 0.078 0.030 CD 0.54 0.57 3.13 0.88 2.449 1.02 A 2 5.51 2.47 0.089 3.53 0.20 3.78 B 2 0.62 0.023 0.86 0.017 0.68 0.38 C 2 0.06 0.34 0.68 1.18 0.64 0.28 D 2 3.466 0.050 3.22 0.033 6.964 0.053 C.V 2.72 21.90 6.16 19.01 6.54 2.45 S.D p=0.05 2.04 0.92 1.34 2.31 1.45 1.94 Note: , significant at P0.05 and P0.01 respectively; Least Squares Means analysis. Table 6. Experimental data for colour attributes using four factor three level Box- Behnken design Extrusion Process Variables Colour Attributes Moisture content Screw speed rpm Die temp °C Ingredient Composition- R:P:C L a b ? E Chroma Hue angle 17 400 150 70:20:10 71.7 5.4 22.5 14.96 23.14 76.50 14 400 120 70:20:10 77.1 2.85 22.2 10.85 22.38 82.68 17 300 150 60:30:10 73.8 4.7 22.5 13.22 22.99 78.20 17 400 150 70:20:10 76.6 3.4 21.05 11.01 21.32 80.82 17 300 120 70:20:10 71.55 4.7 21.25 15.48 21.76 77.53 14 400 150 60:30:10 75.7 5.1 23.4 11.58 23.95 77.70 17 400 150 70:20:10 73.6 4.35 20.6 11.99 21.05 78.08 20 300 150 70:20:10 74.8 4.25 22.15 11.07 22.55 79.14 17 500 150 60:30:10 73.75 5.4 23.9 16.55 24.50 77.27 17 400 120 80:10:10 74.35 3.4 19.45 11.04 19.74 80.08 17 300 150 80:10:10 76.75 2.9 19.4 7.6 19.62 81.50 20 400 180 70:20:10 75.15 3.6 20.75 12.95 21.06 80.16 20 400 150 60:30:10 77.15 3.2 21.15 9.92 21.39 81.40 20 400 120 70:20:10 75.4 3.85 20.75 11.05 21.10 79.49 17 300 180 70:20:10 73.3 4.85 22.9 13.32 23.41 78.04 14 400 150 80:10:10 75.45 5.05 22.4 13.37 22.96 77.30 17 400 120 60:30:10 74.6 4.85 21.8 14.75 22.33 77.46 17 500 180 70:20:10 77.65 4.55 22.7 13.54 23.15 78.67 17 500 120 70:20:10 74.15 4.75 23.55 13.99 24.02 78.60 20 400 150 80:10:10 74.95 2.65 18.65 9.38 18.84 81.91 17 400 180 80:10:10 70.95 5.35 21.55 15 22.20 76.06 14 400 180 70:20:10 77.9 4.85 22.8 9.8 23.31 77.99 17 500 150 80:10:10 76.8 3.85 21.6 9.2 21.94 79.89 20 500 150 70:20:10 77.5 1.35 17.75 9.12 17.80 85.65 14 500 150 70:20:10 78.1 4.35 22.2 11.53 22.62 78.91 17 400 180 60:30:10 74.2 5.4 24.05 14.36 24.65 77.35 14 300 150 70:20:10 75.45 5 24.35 11.76 24.86 78.40 110 Journal of Food Legumes 263 4, 2013 speed of 450 rpm for the samples having moisture content upto 18 whereas lowest L-values were attained by the combination of low screw speed below 350 rpm and higher moisture content above 18. The ingredient composition had a non- significant effect on the L-value of extruded samples. The die temperature also had an influence on L- value with lower die temperatures led to lower L-values and vice–versa. Thus, highest L-values were obtained by the combination of higher screw speed, die temperature and lowest moisture content. The a-values for extrudates ranged from 1.35 to 5.4 Table 6. The a-value was significantly P0.05 influenced by moisture content Table 5. The a-value decreased consistently with increasing moisture content. Since the red lentil gives an yellow colour to the sample after extrusion cooking, the b-value ranged from 17.75 to 24.35 Table 6. The moisture content and ingredient composition had a significant P0.05 influence on b-value of flour. Whereas, the screw speed had a non-significant effect on the b-value of extruded samples Table 5.The highest b-values were observed in samples having higher percentage of red lentil in sample and lower moisture content. The higher b- values were recorded below the moisture content of 15. On the other hand, the ingredient composition also had a significant P0.05 impact on b-values. The highest b-values were recorded for the composition of 60: 30:10 and vice-versa. This can be easily explained by the high ratio of red lentil in the composite flour which gave yellow colour to the samples and increased b-value of the product. The screw speed did not influence the b-value of extruded samples. The chroma value for extruded samples ranged from 17.80 to 24.86 Table 6. Chroma value was significantly P0.05 influenced by moisture content and ingredient composition Table 5. The highest value of chroma was recorded for samples at lowest moisture content having lowest percentage of rice flour and vice- versa. These results can be directly related to the influence of total moisture and ratio of red lentil on the samples. Screw speed and die temperature had a non- significant effect on the chroma value of extrudates. Chroma was also influenced by the die temperature. The highest value of chroma was recorded above the die temperature of 170°C. On the other hand, the screw speed had almost no influence on the chroma values of the extruded samples. The values for hue angle for the extruded samples varied from 76.05 to 85.65 Table 6. The hue angle was significantly P0.05 influenced by the moisture content of the extruded samples Table 5. The highest hue values were recorded above the moisture content of 18. The screw speed, die temperature and composition had a non- significant effect on the hue angle of extruded samples. The lowest hue angle was obtained in the samples having screw speeds below 420 rpm. Also, higher die temperatures led to lowest hue angle in extruded products and vice- versa. The ingredient composition also influenced the hue angle of the extruded products developed with the ingredients lowest values being recorded for proportion with rice component below 70 i.e. 70R:20P:10C. Texture Profile Analysis TPA Fig 5. Contour plots depicting the effect of process parameters on hardness N Hardness: The hardness for extruded samples indicated a variation from 59.89 to 194.31 N Table 8 with significant P0.05 influence of moisture content, screw speed and die temperature on overall hardness quality of extrudates Table 7. It is clear from fig 5 that the hardness of the extruded product decreased with the increase in the moisture content. At a given value of 300 rpm screw speed, the hardness of the extruded product decreased from 154.29 N to 111.69 N with the subsequent increase in the moisture content from 14 to 20. The hardness also indicated an increase with the increase in the screw speed of the extrudates. At a given moisture content of 14, the hardness of the sample increased from Alam et. al. : Optimization of extrusion process variables for development of pulse-carrot pomace incorporated rice based 111 Textural Quality Attributes F values Extrusion Process Variables Hardness Adhesiveness Springiness Cohesiveness Gumminess Chewiness Resilience Moisture Content A 5.25 0.99 6.99 3.30 3.47 2.76 3.12 Screw Speed B 10.45 0.40 0.11 6.92 0.57 0.53 4.02 Die Temperature C 5.78 0.86 0.084 5.93 0.041 1.14 0.27 Ingredient Composition-R:P:C D 34.25 0.076 21.08 4.34 3.45 7.54 4.06 AB 0.35 6.37 6.62 6.57 0.68 2.49 10.93 AC 0.29 3.46 8.62 4.64 1.48 1.89 3.77 AD 0.64 1.15 1.70 5.48 6.28 7.27 5.22 BC 1.70 8.89 0.11 0.17 0.30 0.51 0.66 BD 3.18 2.43 0.66 0.33 0.071 0.60 0.60 CD 1.56 6.70 4.44 0.56 1.95 1.93 0.14 A 2 0.041 1.12 19.86 0.38 0.21 1.78 0.57 B 2 6.10 4.11 6.46 0.20 2.84 0.085 0.69 C 2 0.82 3.08 18.42 12.31 26.51 11.26 7.96 D 2 10.37 1.30 27.06 3.16 16.96 2.57 2.76 C.V 11.71 225.12 9.55 36.46 41.77 50.98 34.03 S.D p=0.05 17.74 7.46 0.027 0.056 1014.79 393.55 0.044 154.28N at 300 rpm to 168.89N at 400 rpm. This value further increased to 169.44N at 500 rpm respectively. Fig 5. clearly indicates that the hardness of extrudates increased with the increase in die temperature while showing a slight decrease at the higher die temperatures. For a given rice percentage of 60 in ingredient composition, the hardness increased from 117.85N to 181.38N with the increase in die temperature from 120 to 180°C. The effect of ingredient composition on hardness of extrudates was non-significant. Table 7. Analysis of variance of extrusion process variables on textural attributes Table 8. Experimental data for textural attributes using four factor three levels Box- Behnken design Note: , significant at P0.05 and P0.01 respectively; Least Squares Means analysis. Extrusion Process Variables Textural Quality Attributes Moisture content Screw speed rpm Die temp °C Ingredient Composition- R:P:C Hardness N Adhesiveness Springiness Cohesiveness Gumminess Chewiness Resilience 17 400 150 70:20:10 149.4067 -0.314 0.3335 0.126 3924.62 637.488 0.112 14 400 120 70:20:10 110.0807 -0.3565 0.23 0.1225 1801.45 640.04 0.1025 17 300 150 60:30:10 173.2098 -0.516 0.2645 0.074 1339.316 370.134 0.0645 17 400 150 70:20:10 194.3099 -24.3815 0.3665 0.269 5279.393 1287.68 0.2395 17 300 120 70:20:10 107.5563 -0.2625 0.222 0.0675 747.448 166.3805 0.0675 14 400 150 60:30:10 179.4418 -0.053 0.2965 0.151 3644.306 1258.282 0.1445 17 400 150 70:20:10 170.0219 -17.842 0.3695 0.2385 5872.374 1702.16 0.1295 20 300 150 70:20:10 125.8026 -0.02 0.3125 0.211 2543.502 772.2165 0.1905 17 500 150 60:30:10 177.9249 -0.3655 0.228 0.0495 908.79 206.97 0.0515 17 400 120 80:10:10 128.3039 -0.2285 0.2105 0.1905 2477.736 777.0685 0.1545 17 300 150 80:10:10 130.811 -0.01 0.272 0.132 987.87 251.964 0.136 20 400 180 70:20:10 149.2116 -16.1585 0.3425 0.36 5538.055 2101.696 0.284 20 400 150 60:30:10 112.5082 -0.776 0.302 0.278 3628.075 1062.306 0.1535 20 400 120 70:20:10 59.89665 -0.517 0.2315 0.1435 892.704 225.1135 0.115 17 300 180 70:20:10 144.449 -0.2635 0.2635 0.095 1299.693 244.246 0.073 14 400 150 80:10:10 185.323 -0.6895 0.2865 0.17 2017.37 726.46 0.1465 17 400 120 60:30:10 115.3241 -0.031 0.2555 0.071 1485.328 404.8765 0.063 17 500 180 70:20:10 175.9571 -0.3505 0.279 0.1315 576.028 794.6505 0.1095 17 500 120 70:20:10 138.4315 -23.6265 0.194 0.0395 563.4055 107.953 0.0365 20 400 150 80:10:10 164.6938 -0.005 0.274 0.25 3111.04 1093.03 0.226 17 400 180 80:10:10 192.2227 -0.7425 0.404 0.283 3821.6 1976.78 0.2305 14 400 180 70:20:10 171.0555 -0.01 0.271 0.076 1359.559 394.714 0.0725 17 500 150 80:10:10 179.869 -0.246 0.3485 0.1915 3393.573 1182.995 0.1555 20 500 150 70:20:10 151.0783 -0.6875 0.2335 0.0675 1127.829 329.99 0.061 14 500 150 70:20:10 161.1556 -0.235 0.3295 0.1885 3207.361 1105.898 0.1505 17 400 180 60:30:10 186.402 -0.3865 0.21 0.0945 1901.415 553.497 0.0735 14 300 150 70:20:10 154.8279 -0.446 0.251 0.09 2149.839 466.951 0.111 112 Journal of Food Legumes 263 4, 2013 Adhesiveness: Adhesiveness values ranged from -0.005 to -24.38 Table 8. Adhesiveness of extruded samples was non-significantly influenced by the process parameters. However, the maximum adhesiveness was observed in the samples maximum moisture content, screw speeds and minimum die temperatures. The ingredient proportions showed non-significant effect on adhesiveness Table 7. Springiness: Springiness ranged from 0.194 to 0.404 in the extruded samples Table 8. The statistical results indicate that springiness was significantly P0.05 influenced by die temperature and ingredient composition used during extrusion processing Table 7. The springiness indicated a significant P0.05 increase with subsequent increase in die temperature with highest value recorded beyond 168°C. This can be directly related to increase in the level of chemical reactions like starch gelatinization with increasing temperature which influence the plastic nature of the product. The flour composition also directly influenced springiness of the sample as springiness increased with decrease in ratio of pulse in sample. But, there was no significant effect of screw speed and moisture on springiness of extruded products. Cohesiveness: Cohesiveness values varied from 0.36 to 0.39 Table 8. The overall effect of different process parameters viz. screw speed, die temperature, moisture content and composition on the cohesiveness of extruded samples was significant P0.05 Table 7. It was statistically evaluated that cohesiveness was significantly P0.05 influenced by moisture content and flour composition. Lower moisture content indicated lower values of cohesiveness in the extruded samples and vice versa. However, the cohesiveness was also directly influenced by higher content of rice flour in the sample as the higher cohesiveness values were recorded for samples containing higher rice contents in their composition. The screw speed and die temperatures had a little influence on the cohesiveness of extruded samples. The higher die temperatures denoted to higher values of cohesiveness and vice versa. Gumminess: The gumminess values of extruded products indicated a large variation from 563.405 to 5872.37 Table 8. The gumminess of extruded products was significantly P0.05 influenced by the combination of process parameters Table 7. The screw speed, die temperature and flour composition influenced the overall gumminess of the product. The gumminess of product increased with increase in die temperature indicating maximum value above the temperature of 180°C. On the other hand, the minimum values were observed below 130°C. The higher moisture content also led to increase in gumminess of the product with highest value recorded above 19 percent. The effect of screw speed and flour composition on the gumminess of extruded products was minimal. However, the gumminess of extruded products increased subsequently with the increase in screw speed and percentage of rice in the flour. Chewiness: The chewiness of extruded products indicated a large variation from 107.953 to 2101.7 Table 8. The chewiness of extruded products was significantly P0.05 influenced the combination of process parameters with large significant P0.05 effect due to die temperature Table 7. The chewiness indicated a large increase with subsequent increase in die temperature with highest value recorded above 162° C. The increase in chewiness due to increase in die temperature can be owed to the increase in more chewi substances due to faster chemical reactions which take place at high temperature. On the other hand, the minimum values were observed below 126°C. The screw speed, moisture content and flour composition influenced the overall chewiness of the product. The higher chewiness values were recorded at higher moisture content and screw speed. The effect of flour composition on the chewiness of extruded products was also pronounced as greater chewiness was recorded for the samples containing rice above 75 and vice versa. Resilience: The resilience values varied between 0.036 to 0.284 Table 8. The effect of process parameters on the resilience of the extruded products was significant P0.05 with greater influence of ingredient composition Table 7. The ingredient composition used had a large influence on the resilience with higher values recorded for the flour containing rice flour above 75. On the other hand, the lower resilience Table 9. Optimum values of extrusion process parameters and responses Target Experimental Range Extrusion process parameters Min Max Optimum value Desirability Moisture content In range 14 20 20 Screw speed rpm In range 300 500 340 Die temperature C In range 120 150 120 Rice flour In range 60 80 60 Pulse flour In range 10 30 30 Carrot pomace flour In range 10 10 10 Responses Predicted values Protein Maximize 7.89 11.74 11.32 0.737 Fiber Maximize 11.7 11.92 11.91 Overall Acceptability Maximize 4.33 8.33 5.45 Hardness N Minimize 59.89 70.00 58.05 Colour difference In range 7.6 16.55 13.36 Alam et. al. : Optimization of extrusion process variables for development of pulse-carrot pomace incorporated rice based 113 values were recorded for flour composition containing rice flour below 65. Moisture content, screw speed and die temperature also influenced the resilience of extruded products. The highest values of resilience were obtained at higher moisture contents and die temperature. The screw speed had a non- significant effect on the resilience of the extruded products. Optimum conditions for ready-to-eat extruded hardness with colour difference in range. These constraints resulted in “feasible zone” of optimum conditions shaded area in the superimposed contour plots fig 6. The optimum ranges of process parameters obtained for development of extrudetes were: 19.47-20 of moisture content, 120 to 125°C die temperature, 333 to 381 rpm screws speed and 60 to 61 of rice flour with 10 carrot pomace in ingredient composition table 9. In order to optimize the process conditions for extrusion process by numerical optimization, which finds a point that maximizes the desirability function; importance of ‘4’ was given to protein and fiber content while importance of ‘3’ was given to all other parameters. The optimum operating conditions for screw speed, die temperature, moisture content and rice content in ingredient composition was 340 rpm, 120°C, 20 and 60, respectively. Corresponding to these values of process variables, the values obtained were 11.32 protein content, 11.91, fiber content, 58.05 N hardness, 5.45 overall acceptability and 13.36colour difference Table 4. The overall desirability, which ranges from zero outside of the limits to one at the goal, was 0.737. It could be concluded from the present study that the response surface methodology is effective in optimizing extrusion process parameters for red lentil-carrot pomace incorporated ready-to-eat rice based expanded product with feed moisture in the range of 14 to 20, die temperature 120- 180°C, screw speed 300-500 rpm and formulation rice flour: pulse flour; 60-80: 10-30 with 10 carrot pomace flour. Graphical techniques, in connection with RSM, aided in locating optimum operating conditions, which were experimentally verified and proven to be adequately reproducible. The optimum process parameters obtained for development of extrudates were 20 feed moisture, 340 rpm screw speed, 120°C die temperature and formulation of 60:30:10; rice flour: pulse flour: carrot pomace flour; for achieving the maximum possible protein content, fiber content, overall acceptability with minimum hardness and colour difference. REFERENCES Adsule RN. 1996. In E Nwoloko and J Smartt Eds., Food and feed from legumes and oilseeds. Chapman and Hall Pub. Pp. 84-110. Agriculture and Agri-Food Canada. 2006. Lentils: Situation and outlook. Bi-weekly Bulletin, 197. Retrieved 21.08.08. Alam MS, Kumar Mahesh, Kumar Satish and Bhandari Arjun 2011. Optimization of process parameters for osmo-mechanical dehydration of shelled peas Pisum sativum. Journal of Food Legumes 243: 218-24. Altan A, McCarthy KL and Maskan M. 2008a. Twin-screw extrusion of barley-grape pomace blends: Extrudate characteristics and determination of optimum processing conditions. Journal of Food Engineering 89: 24-12. Altan A, McCarthy KL and Maskan M. 2008b. Evaluation of snack food from barley-tomato blends by extrusion processing. Journal Fig 6. Overlaid contours of different responses for optimization of extrusion process parameters product: Graphical multi-response optimization technique was adopted to determine the workable optimum conditions for the development of extruded product using design expert software Stat ease, DE 8.0.6.1. The contour plots for all the responses were superimposed and regions that best satisfied all the constraints were selected as optimum conditions. The main criteria for constraints optimization were maximum possible protein, fiber and overall acceptability and lower 114 Journal of Food Legumes 263 4, 2013 of Food Engineering 84: 231-242. Amerine MA, Panngborn RM and Roessler EB. 1965. Principles of Sensory Evaluation of Food. Pp 5. Academic Press, London. AOAC 2005 Official Methods of Analysis of AOAC International. Pp 20-35. 18 th ed. USA Bohm V, Otto K and Weissleder F. 1999. Yield of juice and carotenoids of the carrot juice production”, In: Symposium Jena-Thuringen, Germany. Pp 115-119. Cochran WG and Cox GM. 1964. Experimental designs. New York: John Wiley Sons, Inc. Asd Ding QB, Ainsworth P, Tucker G and Marson H. 2005. The effect of extrusion conditions on the physicochemical properties and sensory characteristics of rice based expanded snacks. Journal of Food Engineering 66: 283-289. Gnanasekharam V, Shewfelt RL and Chinnan MS. 1992. Detection of colour differences in green vegetables. Journal Food Science 57: 149-54. Grigelmo-Miguel N and Martín-Belloso O. 1999. Comparison of dietary fibre from by-products of processing fruits and greens and from cereals. LWT – Food Science Technology Pp. 503-508. Kadan RS, Bryant RJ and Pepperman AB. 2003. Functional properties of extruded rice flours. Journal of Food Science 68: 1669-1672. Kumar Mahesh, Alam MS, Kumar Satish and Singh Jarnail. 2009. Textural degradative kinetics of selected legumes of Punjab. Journal of Food Legumes 224: 291-295. Montgomery DC. 2001. Design and Analysis of Experiments. New York Wiley. Pp. 416-419. Moscicki L, Mitrus M, Wojtowicz A, Technika ER. 2007. PWRiL, Warszawa Nawirska A and Kwasniewska M. 2005. Dietary fibre fractions from fruit and vegetable processing waste. Food Chemistry 912: 221- 225. Ng A, Lecain S, Parker ML, Smith AC and Waldron KW. 1999. Modification of cell wall polymers of onion waste. III. Effect of extrusion-cooking on cell wall material of outer fleshy tissue. Carbohydrate Polymers. 39: 341-349. Ranganna S 1997 Handbook of Analysis and Quality Control for fruit and vegetable Products. 2nd edn. Pp 1112. Tata Mc.Graw Hill Pub.Co.Ltd., New Delhi, India. Serena A and Bach Knudsen KE. 2007. Chemical and physicochemical characterization of co-products from the vegetable food and agro industries. Animal Feed Science and Technology. 139: 109–124. Stojceska V, Ainsworth P, Plunkett A, Ibanoglu E and Ibanoglu S. 2008. Cauliflower by-products as a new source of dietary fibre, antioxidants and proteins in cereal based ready-to-eat expanded snacks. Journal of Food Engineering 87: 554-563. Yagci S and Gogus F. 2008. Response surface methodology for evaluation of physical and functional properties of extruded snack foods developed from food-by-products. Journal of Food Engineering 86: 122-132. Journal of Food Legumes 263 4: 115-119, 2013 Area expansion under improved varieties of lentil through participatory seed production programme in Ballia District of Uttar Pradesh S. K. SINGH, RIYAJUDDEEN, VINAY SHANKAR OJHA and SANJAY YADAV Indian Institute of Pulses Research, Kanpur – 208024,Uttar Pradesh, India; E-mail : sushilsinghiipryahoo.co.in Received : April 02, 2013 ; Accepted : August 12, 2013 ABSTRACT Attempts have been made through a pilot project to introduce lentil crop in rice-fallow of Ballia district of eastern U.P. to break the mono-cropping and to introduce improved varieties . A block Sohown was selected and data were collected from the respondent through PRA survey and structured interview schedule. The socio-economic status is related to adoption of recommended package of practices for cultivation of lentil, opinion of the farmers and impact of the project. Maximum 55.83 farmers adopt 2 solution of urea improves the plant growth as well as yield . Almost all the farmers used recommended seed rate 16 kgacre 40 kgha of lentil after two years of participatory lentil production programme. NDL-1 variety of lentil performs better and PL-6 variety has been chosen as short duration 105-115 days under monocropped rainfed farming situation. Participatory Varietal Selection Trials are beneficial for selection of best performing variety in a particular farming situation. Efforts made to Register farmers association under “Uttar Pradesh society Registration Norms- 1860” in the project implementing area for the production of quality seed on large scale through close linkage with National Seed Corporation, Seed Certification Agencies, Private dealers and local traders. Thus farmers should be trained properly about improved production technology of lentil and these trained farmers would be able to educate and transfer the technology more electively among the lentil growers. Productivity enhancement, nutritional security and rural livelihood could be achieved through participatory farmers seed production programme. Key words: Farmers Association, FPVSTs, Impact, Lentil,, PRA Lentil Lens culinaris is one of the major rabi pulse crops grown in India since time immemorial and contributes significantly to food, feed and sustainable farming systems. In India, lentil is grown in an area of 1.48 million hectare with a total production 1.03 million tons with an average yield 697.00 kg ha. Recent estimates suggested that approximately 11.65 million hectare area is under rice-fallow in the country. 0.35 million hectare area in UP as a rice-fallow providing ample scope for expansion of lentil crop in the state. Farmers must use pure and healthy seeds which have slandered germination percentage. The high quality seed are those which have genetic purity, physical purity, health standards, germiniability and moisture percentage in accordance with the minimum seed certification standards. Hence the farmers can increase approximately 20 production by use of quality seed of lentil. Unfortunately, this could not happen because of farmers lack of awareness about improved varieties and matching production technologies, inadequate supply of seed of appropriate varieties. The farmer does make arrangements of many inputs but the seed is the chief input among other inputs. To promote the improved varieties and production technologies of lentil in rice-fallow the participatory seed production has been initiated, developed as a community based seed production approach for a sustainable seed supply for farmers, it is an approach of producing and distributing seed with the participatory involvement of farmers group. In this approach, seed producer farmers associations are formed to multiply the seed of farmer- preferred varieties using a cost effective approach having several unique features. Thus helping to expand area under lentil and also improve the productivity by way of augmenting the supply of quality seeds and popularizing the refined package of practices it takes account of the entire seed innovation system from initial identification of new varieties through participatory varietal selection through to commercial seed production. The good quality of seed true to its type, free from ad mixture of other variety seed, having high percentage of germination and free from seed born diseases. Hence keeping these variables in mind, farmers should be trained properly about improved production technology of lentil and these trained farmers would be able to educate and transfer the technology more effectively among the farmers of neighboring villages. RESEARCH METHODOLOGY DAC-ICARDA-ICAR collaborative project “Enhancing lentil Production for Food, Nutritional Security and Improved rural Livelihood” is being implemented since October 2010. Sohown block is the project site of the Ballia district the total 09 village where lentil is grown in large scale were selected for gathering information. Base line Survey: A baseline survey was conducted in 2010 in partner villages of Sohown block of Ballia districtto assess the situation from production to marketing.The major steps used in conducting base line survey are as follows- ● Semi structured interviewwere conducted in the villages with active involvement of farmers. ● PRA survey was conducted in the village with the group of farmers Participatory mapping, Transect Walk, 116 Journal of Food Legumes 263 4, 2013 scoring, Matrix Ranking, change and trends. ● Collection of data from farmers mainly related to socio- economic, adoption of improved package of practices, farmers opinion and constraints in seed production. ● Collection of Secondary data of the selected villages from the office of District Agriculturelekhpal. The data collected were processed, summarized and tabulated for statistical analysislike percentage, average and scaling for meaningful interpretation of results. Farmers Participatory Varietal Selection Trial FPVST: Farmers Participatory Varietal Selection Trials has helped in building of confidence among farmers for promoting as well as ensuring seed sufficiency at village level. Farmers participatory Varietal Selection Trial son farmers field with four improved varieties NDL-1, HUL-57 PL-6 and WBL-77 alongwith local variety was conducted to identify farmer- preferred varieties. Each FPVST was conducted in an acre. The varieties were evaluated for seed yield and other economic parameters besides taking into consideration the farmers perception on their performance. Seed Production: To as certain farmers preferred as well as shifting of area under improved varieties of lentil was sown in normal condition in first week of November whereas late variety were sown after second fortnight of November to first week of December with recommended Seed rate 16-20 kg acre. Before the start of cropping season, farmers meeting were organized to decide our seed production programme. Following activities were also undertaken to develop farmers capacity in quality seed production, processing and marketing by facilitating them to form their association. 1 Farmers Training in Crop Management and Seed Production: To ensure seed sufficiency at village level efficient and effective quality seed production of lentil farmers were trained in various technological aspects on crop production and seed production included varietal description, seed treatment, nutrient management, Insect-pest and disease, Isolation distance, roguing in seed production fields and crop harvesting technology etc. 2 Close Linkage with Formal Seed Sector: The selected improved varieties growers were linked with Uttar Pradesh State Seed Certification Agency, Mau for seed certification to strengthen formal Seed sector for lentil Seed Production. KVK were also linked with farmers to provide them day to day latest technical information and ensure quality of seed produced. 3 Formation of Registered Farmers Association: For Promotion of Formal and Informal seed system two registered farmers association has been formed to ensure the seed sufficiency of farmers preferred variety of lentil for multiplication of seed at village level.”Madaura Kisan Sewa Samiti” 25 active member and “Jai Vigyan Kisan Sewa Samiti” 18 active memberformed underthe “Uttar Pradesh Society Registration Norms -1860” in 2012. RESULTS AND DISCUSSION Socio-economic characteristics of the Lentil Seed Producers: The table-1 showed the socio-economic status of the farmers that 10.00 farmers are illiterate and 41.66 are having graduation and above level of education. Maximum 51.67 farmers are having 5-8 members in his family and 40.00 farmers were having joint family. Majority of 41.67 farmers having 5-6 ha of land holding whereas 15.83 farmers comes under the category of less than 2 ha area. Due to large land holdings 94.17 farmers used tractor drawn seed drill in line sowing. Majority of farmers 55.83 depends on tube wells for irrigation. Table-1 Socio-economic Status of the Farmers of District Ballia Sl. No. Particulars No. of farmers Percentage 1. Educational Status- Illiterate Primary Middle High School Intermediate Graduation Post-Graduation 12 07 10 22 19 43 07 10.00 05.83 08.33 18.33 15.83 35.83 05.83

2. Size of Family-