TIME COURSE CHANGES IN GROWTH AND PHYSIOLOGICAL PARAMETERS OF AJWAIN

TIME COURSE CHANGES IN GROWTH AND PHYSIOLOGICAL PARAMETERS OF AJWAIN (Trachyspermum ammi L.) UNDER DROUGHT STRESS
SAIMA RASHID1, ABDUL WAHID2
1Muhammad Nawaz Shareef University of Agriculture,Multan
2University of Agriculture Faisalabad, Faisalabad

Abstract
Drought stress is a great deterrent to the plant growth and productivity. In this study the effect of drought stress was observed on the growth and metabolic attributes of Ajwain (Trachyspermum ammi L.) at various time intervals. Exposures to drought stress severely affected different morphological parameters such as shoot and root length, shoot and root dry weight. Among the other metabolites, soluble sugars, proline and total free amino acids indicated an increase at all harvests. Anthocyanin was accumulated under drought stress, but soluble phenolics were declined. These compounds act as compatible solutes and thus, their accumulation within plant body appeared to protect the plant possibly against the damaging effects of water stress. Drought stress triggers different systems which minimize the damages caused by oxidation. Level of antioxidant ascorbic acid was found to increase under shortage of water. In conclusion Ajwain showed sensitivity to drought stress at various stages, possibly due to reduced accumulation of osmoprotectants and important secondary metabolites such as soluble phenolics.
Key words: Ajwain, drought

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Introduction
Land plants are non motile organisms that are unable to move from one place to another place under unfavorable conditions. Since they stay on one place throughout their life, so they face different types of biotic and abiotic stresses (Bhatti et al., 2013). Stress is any factor that interferes natural plant mechanism. Plant is unable to manage its natural growth under stress and utilize extra amount of energy to minimize the negative effects of stress. (Asadi et al., 2012).
Naturally, plants are bared to various biotic and abiotic pressures. Among abiotic stress, water scarcity is one of the most adverse stress. It affects normal plant growth and development (Anjum et al., 2011). Water scarcity affects crop efficiency more than any other abiotic stress (Shao et al., 2009). Drought acts as multidimensional pressure damaging plant growth at different developmental phases. It is not easy to construe the effects of water stress at different developmental stages of a plant or whole crop in one field due to a variety of plant behaviors towards drought (Majeed et al., 2011).
It is ascertained that shortage of water is very dangerous particularly at the initial stages of seed germination, seed growth and establishment of seed. Solute regulation plays a vital role in maintaining plant growth and development under drought stress. Different growth stages of a plant have different response towards water stress. This behavior of the plant depends upon severity and length of water scarcity as well as the type and developmental stage of the plant (Shitole and Dhumal, 2012).
Favorable growth environment is necessary for normal plant growth and development which determine plant vigor and productivity and all this is significantly influenced by water accessibility (Gan et al.,2004). The most prominent and severe impacts of water stress can be seen during reproductive stage and grain filling stage of plant growth. Water stress applied during flowering stage tends plant to shortly complete its life cycle, thus reducing the crop productivity (Sinaki et al.,2007).
Materials and methods
Experimental details
Under the influence of stress of drought on carom seeds, the present study was conducted to carry out the effect of drought on the morphological and physiological parameters of carom seeds. The experiment was laid out in Randomized Complete Block Design (RCBD) in triplicate. Carom seed plants aged two months were drought-stressed by halting the irrigation water supply. The data were recorded on fortnightly basis for about four times to determine the growth and physiological characters. Half of the samples were deep frozen at -30ºC for fresh analysis, while the other half were oven dried. Frozen fresh sample were used for physiological parameters.
Determination of Growth attributes
Shoot length and root length were measured after harvesting the plants. However, for the measurement of dry weight, the shoot and root parts were dried in an oven at 70oC for a week.
Determination of Physiological Parameters
Determination of Ascorbic acid
Extract leaf material (0.25 g) with 10 ml 0f 6% Trichloroacetic acid. Mix 4 ml of extract with 2 ml of 2% Dinitrophenyl hydrazine. Followed by addition of 1 drop of 10% thiourea (in 70% ethanol). Boil the mixture for 15 min in water bath and then cool in ice. When at room temperature add 5 ml of 80% H2SO4 at 0 ºC and note the absorbance at 530 nm (Mukherjee and Choudhari, 1983).
Determination of soluble phenolics
Soluble phenolics were determined with the method of Jukenen-Titto (1985). For this purpose, 50 mg of ground fresh material was crushed and extracted in 1 ml of 80% acetone at 50ºC for 1 h and samples were centrifuged at 12000 x g for 15 min. a 100 µl aliquote of the supernatant was diluted with distilled water to 1 ml in a 10 ml capacity test tube and 0.5 ml of folin-ciocalteu’s phenol reagent was added followed by vigorous shaking. After that 2.5 ml of 20% Na2CO3 was added and made the volume to 5 ml and vortexed vigorously for 5-10 second, waited for 20 min and measured the absorbence at 750 nm using a spectrophotometer with 80% acetone as blank. Total values of tannic acid equivalent soluble phenolics were computed from the standard curve prepared from 20, 40, 60, 80 and 100 µg of Tannic acid (prepared from 1000 µg ml stock).
Free proline
For this purpose, the method of Bates et al. (1973) was followed. A 0.5 g of seeds was homogenize in 10 ml of 3% sulphosalisylic acid and filtered the homogenate through whatman No. 2 filter paper, Two ml of filtrate was add to 2 ml of acid ninhydrin and 2 ml of glacial acetic acid in a test tube. Heat the mixture at 100oC in a water bath for I h and terminate the reaction in icebath. Extracted the reaction mixture with 4 ml of toluene after vigorously vortexing for 15 to 20 sec. Aspirated the chromophore containing free proline in a test tube, warmed to room temperature and measured the absorbance at 520 nm. The above procedure was followed for blank using 2 ml of aqueous sulphosalisylic acid.
Total free amino acids
Total free amino acids were determined with the method of Hamilton and Van-Slyke (1973). Fresh material was extracted by heating in water. Then 1 ml of extract was taken in 25 ml test tube, added 1 ml of 10 % pyridine and 1 ml of 2 % ninhydrin solution in each tube. Then heated the tubes in boiling water bath for about 30 min and made the volume in each tube to 50 ml with distilled water. The optical density of the colored solution was read at 570 nm using spectrophotometer. Developed a standard curve with Lucine and calculated free amino acid by the formula given below:
Total amino acid (mg/g Fresh weight) =
Graph reading of sample x volume of the sample x dilution factor
Weight of the tissue x 1000
Determination of anthocyanins
A 0.1 g of fresh material was extracted with 1ml of acidified methanol (1% HCl, v/v). Heated at 50 ºC for 1 h and filtered. Absorbance of the filtrate was measured at 535 nm on a spectrophotometer. The background was set with acidified methanol. The amount of anthocynins was given as A535.
Soluble sugars
To do this, presoaked seeds (0.1 g) were boiled in 5 ml of distilled water for 1 h. Filtered and made the volume up to 50 ml with distilled water. Then 1 ml of the extract was added to 5 ml of anthrone reagent along the side wall of the test tube and briefly vortex. Heated the mixture in water bath at 90-95oC for 20 min. Cooled and took the absorbance at 620 nm using a spectrophotometer
Results and Discussions
In the present study, the data was collected for various growths, physiological and yield attributes, which are described below under separate sections.
Table 1: Analysis of variance (mean squares) of the variance sources of the ajwain plant grown under drought stress at a various harvests.
Parameters Sources of variations
Treatments (T)
DF = 1 Harvests (H)
DF = 3 S ? N
DF = 3 Error
DF = 16
Shoot length 50.94** 8.87** 4.74ns 4.17
Root length 0.40ns 171.71** 10.35** 0.94
Shoot dry weight 2.86** 1.02** 0.29** 0.005
Root dry weight (×10-3) 0.09** 0.01** 0.04** 8.78
Significant at ** P