N 160 mg.kg–1), Phosphorus (P 20, P 40, P 80 and P 160 mg.kg–1) and Potassium (K 40, K 80 and K 160 mg.kg–1) in the form of Urea (46%, H2NCONH2), Superphosphate (16%, P2O5) and Muriate of potash (60% K2O) respectively. A significant increase in the chlorophyll content was recorded with all the applications of N, P&K. Root protein and carbohydrate contents were found linearly increase with K treatment while a slight decline was found with the higher dosage of N. Root sapogenin content was 1.66, 1.87&1.75 folds higher than the control with N, P, & K respectively. Application of Phosphorus was found to be best for growth and biochemical contents of root tuber.
The genus, Asparagus comprises 150 species (Drost 1997) and consists of herbs, shrubs and vines which are widespread in the Old World and represents highly valuable plant species having therapeutical and nutraceutical importance in addition to being consumed as food (Shasnay et al., 2003). Asparagus racemosus (locally known as Shatavari) is one of the important medicinal plant extensively used by the traditional practioners in India for its medicinal value. The leaves and the tuberous roots of Asparagus are medically important to cure minor to severe disease. Asparagus racemosus is distributed throughout tropical Africa, Java, Australia, India, Srilanka and Southern parts of China (Kirtikar and Basu, 1985). The healing qualities of Shatavari are useful to a wide array of ailments. Being a rasayana or rejuvenating herb, its restorative action is beneficial in woman's complaints. The roots of plant have been referred as galactogogic, bitter-sweet, emollient, cooling, nervine tonic, constipating, aphrodisiac, diuretic, carminative and antiseptic (Chaudhary and Kar, 1992). Roots of A.racemosus were found to possess antioxidant and anti-ADH activity (Kamat et al., 2000 and Wiboonpun et al., 2004), antitumour and anticancer activity (Senna et al., 1993; Shao et al., 1996; Dhuley, 1997 and Diwanay et al., 2004), anti-ulcerogenic activity (Datta et al., 2002), anti-inflammatory activity (Mandal et al., 1998) and antimicrobial activity (Mandal et al., 2000).
Most of the herbal medicine available is derived from the roots of the plant. The commercial success largely depends on quality and yield of root, which is the product of commerce. Among factors responsible for the low yield is low soil fertility, as most tropical soils are deficient in essential nutrients particularly N and P (Jones and Wild, 1975). Poor availability of nutrients in soluble form in the arid and semi-arid soils is the most important limiting factor as compared to that of moist areas. It has been suggested that organic manure should be used in place of chemical fertilizer to avoid long term negative effects of chemical fertilizers on the soil (Parr etal., 1990). However, organic manure is usually required in large quantity to sustain crop production and may not be available to the small-scale farmers (Nyathi and Campbell, 1995), hence the need for inorganic fertilizer. The positive effect of the application of inorganic nutrients on the crop yield and yield improvement has been reported (Carsky and Iwuafor, 1999). Application of N, P and K has been reported to increase the growth and productivity of several plants of arid and semi-arid regions (Kumar et al., 1995). Buries (1959), stated that nitrogen has stimulating effect on root activity and rooting pattern of the crop. It has also been reported available nitrogenous compounds allowed seedling to make a good start. Phosphorus is a major component of important metabolic structure involved in energy utilization and storage mechanism. This is also essential for carbon metabolism which increases the biomass production, its partitioning and ultimately the yield of crop plants (Blevins, 1994). Application of Potassium in different forms has also found to influence plant yield and its chlorophyll contents (Chapagain and Wiesman, 2004).
Balanced fertilizer nutrients can also play a vital role in sustaining high yield of medicinal plants as well as maintaining fertility status of soils on long-term basis. The present investigation was undertaken with nitrogen, phosphorus and potassium to investigate their effects on growth and biochemical contents of root tubers of Asparagus racemosus Willd.
MATERIALS AND METHODS
The study was conducted at the medicinal plant nursery, Department of Botany, University of Rajasthan, Jaipur during August, 2004 to January, 2005. 3-month-old healthy and uniform sized seedlings of Asparagus racemosus Willd. were taken for the present study. Ten replicates were taken for each set of experiment and were conducted in 30 cm earthen pots filled with approximately 4 kg soil (soil: silt: clay, 1:1:1). Plants were irrigated to 50-60 per cent of the field capacity. Different concentrations of Nitrogen (N 20, N 40, N 80 and N 160 mg.kg–1), Phosphorus (P 20, P 40, P 80 and P 160 mg.kg–1) and Potassium (K 40, K 80 and K 160 mg.kg–1) were employed in the form of Urea (46%, H2NCONH2), Superphosphate (16%, P2O5) and Muriate of potash (60% K2O) respectively. The amount of N, P, and K was calculated in these compounds on the basis of available Nitrogen, Phosphorus and Potassium respectively. Nutrients were properly mixed with 4 kg of soil on the blotter paper and then transferred to the 30 cm earthen pots. Plants were harvested in general by uprooting. Fresh weight of aboveground and underground parts was taken just after harvest. The length of shoot and root tubers was also measured. Dry weight was determined after drying the plants in oven at 60 ± 2 °C, till the weight became constant. The dried samples were ground by grinding machine and stored in paper bags for biochemical analysis. Chlorophyll content in fresh leaves was estimated following the method of Arnon (1949) using 80% acetone and absorbance was read at 663 nm and 645 nm. Carbohydrate contents were estimated following Roe (1955) method using Anthrone reagent. Protein contents were estimated following Bradford (1976) and Sapogenin contents were analyzed following method described by Bhagat and Jadeja (2003) using Liberman – Burchard reagent. Yield-contributing parameters including height, fresh and dry weight of both aboveground and underground parts of the plant was also recorded. The data in table 1, 2 & 3 is represented as mean ± SEM (Standard error of mean) of five replicates.
RESULTS AND DISCUSSION
Different forms of inorganic fertilizer in different dosages had a significant effect on all the tested biochemical contents of Asparagus racemosus (Willd.). Yield-contributing parameters were also significantly influenced.
Effect of Nitrogen: N favours maximum chlorophyll biosynthesis. Highest concentration of N (160 mg.kg–1) increased the chlorophyll content two fold (3.119 mg.g-1) over the control plants. All other biochemical contents were also increased significantly with the N treatment compared to control plants (Table 1). A gradual increase in carbohydrate (69.35 mg.g-1), protein (10.83 mg.g-1) and sapogenin (0.313 mg.g-1) contents was recorded with the increasing concentration of N upto 80 mg.kg-1, which also favoring by maximum biomass accumulation 29.25% and 12.44% in both aboveground and underground parts respectively. Application of 80 mg.kg-1 N was found more effective for development of root tuber of Asparagus as observed in present study is in the relation with the findings of Hossain et al., (2006) and Krarup et al., (2002). They found that 50 kg N ha-1 was enough rather than applying higher concentration of nitrogen such as 100, 150 and 200 kg N ha-1. This is due to the fact that higher concentrations of N ion in the soil limit the uptake of other essential macro and micronutrients by the plant. Paschold et al. (1999) reported that excessive N supply can result in less vigorous spears of Asparagus while N deficiency reduces quality. A slight decline in shoot length was recorded with 20 mg.kg–1 concentration of N, but no inhibitory effect on shoot biomass was observed. Influence of N on photosynthetic capacity of plants may be due to the content of activated RUBISCO (Jia and Gray, 2004). Nitrogen is the most important inorganic nutrient in plants and major constituents of proteins, nucleic acids, many cofactors, and secondary metabolites (Marschner, 1995). NO3- addition also modifies resource allocation, growth and development by modulating root-shoot allocation (Scheible et al., 1997 and Stitt and Krapp, 1999) and promoting flowering and tuber initiation (Marschner, 1995).
Effect of Phosphorus: Super phosphate, as the source of P showed significant promotory effect on plant growth and its biomass over the control plants. Chlorophyll concentration was significantly increased with all the concentrations of P and found maximum (2.905 mg/g) in plants given 160 mg.kg-1 P. A linear increase was recorded in the carbohydrate and protein contents with the increasing concentration of P. Maximum carbohydrate (70.17 mg.g-1) and protein (10.69 mg.g-1) contents were recorded at 160 mg.kg-1 P and sapogenin contents were also found to be higher (0.352 mg.g-1) at the same concentration. Phosphorus is most important nutrient element for improving photosynthetic rates, which depends on several factors including nutrient supply (Bisht and Chandel, 1991). Height of plant was also influenced positively with P application, supported by its biomass (Table 2). P supply can modulate the content of activated RUBISCO either directly or indirectly (Usada and Shimogawara, 1991; Rao and Terry, 1995 and Pieters et al., 2001) and influencing photosynthetic activity of plant.
Effect of Potassium: Although Rajasthan soil are not significantly deficient in Potassium but the addition of K as muriate of potash upto the level of 160 mg.kg-1 increased chlorophyll concentration (2.604 mg.g-1) which resulted in increased sapogenin contents (0.329 mg.g-1) in root tubers over the control plants (0.188 mg.g-1) (Table 3). However, maximum chlorophyll a : b ratio was recorded in control plants. It has been studied that Potassium in different forms influenced the plant yield and its chlorophyll contents (Chapagain and Wiesman, 2004) also. Carbohydrate (66.81 mg.g-1) and protein (10.37 mg.g-1) contents were also found maximum at 160 mg.kg-1 K.
The endogenous level of plant growth regulators also affects all the uptake and utilization of minerals. The active principles or precursors are synthesized in the leaves, translocated, biosynthesised, and stored in root tubers. Growth of leaves and development of root tuber depend on several factors such as nutrition (Rethinam et al., 1994). A large proportion of leaf photosynthates are required for the growth and development of tuberous root. The rate and amount of photosynthate produced by the leaves and the proportion of photosynthate that is translocated greatly influence size, yield, development and growth of tuber as well as secondary metabolite accumulation. This transport and partitioning of leaf assimilate to the sink tuber is one of the important factors controlling productivity. However from the present study, we concluded that treatment with N P K fertilizer could improve the biochemical status of the plant. Chlorophyll content was found to be improved best with the application of nitrogen at its highest tested concentration and medicinally important saponins are improved with Phosphorus application. Steroidal saponins are a diverse class of secondary metabolites that are structurally constructed of aglycones (Sapogenins) and sugars (glucose / rhamnose).
A. racemosus contains several steroidal saponin glycosides i.e. Shatavarin I-IV. I, II and IV are derived from a common aglycone moiety, Sarsapogenin. An appropriate hydrolysis of saponin yield sugars and aglycone moiety sarsapogenin. A correlation between sugar and sapogenin contents was observed in the present study.
Highest root growth in terms of % dry weight was observed in plants treated with P, which was further supported by its root sapogenin content. Increase in root growth in all the tested plants is closely associated with biochemical attributes. Compared to control plants root sapogenin content was 1.66, 1.87 & 1.75 folds higher with N, P, & K respectively. Positive correlation between root growth and these parameters could possibly be used for production of high quality plants, as root tubers are the commercial product of interest. Also the estimation of these biochemical attributes under different treatments is informative for finding the best fertilizer nutrition required for the production of its active ingredient. Proteins, carbohydrates, chlorophyll being the direct gene product reflect the genomic composition of cultivars accurately and therefore ideal for finding distinctness. The study therefore has provided NPK fertilization requirement for best growth of such a potential plant commonly used in herbal medicine. The result of this study also showed that there might be differential genotype response to different fertilizer applications, hence the need to determine the fertilizer requirement of the individual genotypes before applying to field production.
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Table 1: Effect of different doses of nitrogen fertilizer on the leaf chlorophyll, root protein, carbohydrate, sapogenin, Plant height and % dry weight of Asparagus racemosus Nitrogen(mg.kg-1)ChlorophyllCarbohydrates (mg.g-1)Proteins (mg.g-1)Sapogenins (mg.g-1)Shoot lengh(cm)Root lengh(cm) AG% Dry weightUG AG : UGControl1.823±0.03853.86±0.2747.23±0.1010.188±0.00825.46 ± 1.3483.66 ± 0.29125.59 ± 0.30210.16 ± 0.0172.518±0.026N 202.810±0.02059.57±0.8089.51±0.3850.225±0.01622.16 ± 1.1664.533 ± 0.20226.81 ± 0.47911.67 ± 0.3512.303±0.107N 403.001±0.03063.74±0.6139.49±0.480.274±0.02338.83 ± 2.1666.766 ± 0.14528.43 ± 0.26612.02 ± 0.1502.364±0.026N 803.038±0.01269.35±0.61610.83±0.2930.313±0.0750.76 ± 0.8689.733 ± 0.40929.25± 0.59012.44 ± 0.2272.353±0.074N 1603.119±0.02566.50±0.48210.29±0.2760.294±0.05644.56 ± 0.9936.866 ± 0.23329.05 ± 0.34711.32 ± 0.6952.582±0.145Fcal388.4849240.4816210.293122.570868.011475.859514.38565.5041.8027Fcrit3.478053.478053.478053.478053.478053.478053.478053.478053.47805Level of Significance ******************* Significant at 5% level of probability, * Significant at 1% level of probability Table 2: Effect of different doses of Phosphorus fertilizer on the leaf chlorophyll, root protein, carbohydrate, sapogenin, Plant height and % dry weight of Asparagus racemosus Phosphorus(mg.kg-1)ChlorophyllCarbohydrates (mg.g-1)Proteins (mg.g-1)Sapogenins (mg.g-1)Shoot lengh(cm)Root lengh(cm) AG% Dry weightUG AG : UGControl1.823±0.03853.86±0.2747.23±0.1010.188±0.00825.46 ± 1.3483.66 ± 0.29125.59 ± 0.30210.16 ± 0.0172.518±0.026P 202.625±0.32361.05±0.518.40±0.1940.229±0.03337.16 ± 0.4405.766 ± 0.4330.32 ± 0.11711.09 ± 0.4572.702±0.072P 402.828±0.02067.22±0.3069.42±0.4350.269±0.02446.83 ± 1.5896.5 ±0.28832.07± 0.35512.44 ± 0.2682.580±0.050P 802.905±0.03969.45±0.36610.07±0.2210.278±0.02548.43 ± 2.7967.066 ± 0.23332.47 ± 0.24212.77±0.1272.452±0.038 P 1602.734±0.03870.17±0.18610.69±0.2010.352±0.03152.76 ± 1.2998.1 ± 0.20832.75 ± 0.16513.36 ± 0.1842.546±0.044Fcal159.5164329.6501269.194421.723748.517430.54267139.082727.70893.5856Fcrit3.478053.478053.478053.478053.478053.478053.478053.478053.47805Level of Significance ******************* Significant at 5% level of probability, * Significant at 1% level of probability Table 3: Effect of different doses of Potassium fertilizer on the leaf chlorophyll, root protein, carbohydrate, sapogenin, Plant height and % dry weight of Asparagus racemosus Potassium(mg.kg-1)ChlorophyllCarbohydrates (mg.g-1)Proteins (mg.g-1)Sapogenins (mg.g-1)Shoot lengh(cm)Root lengh(cm) AG% Dry weightUG AG : UGControl1.823±0.03853.86±0.2747.23±0.1010.188±0.00825.46 ± 1.3483.66 ± 0.29125.59 ± 0.30210.16 ± 0.0172.518±0.026K 402.227±0.03859.82±0.5678.84±0.7840.259±0.03229.16 ± 1.6415.83 ± 0.44027.129 ± 0.17912.28 ± 0.1452.068±0.149K 802.073±0.04063.61±0.4419.71±0.6160.248±0.00830.83 ± 1.1668.2 ± 0.37827.99 ± 0.18412.80 ± 0.3182.307±0.158K 1602.604±0.04066.81±0.59610.37±0.610.329±0.02856.76 ± 1.1058.7 ± 0.43528.516 ± 0.22313.07 ± 0.2252.182±0.045Fcal67.14838207.5674237.616118.0188115.548535.256131.449740.12252.9665Fcrit4.066183.478053.478053.478054.066184.066184.066184.066184.06618Level of Significance *************** ** Significant at 5% level of probability, * Significant at 1% level of probability