“LS – An International Journal of Life Sciences”
Editorial Board EDITOR-IN-CHIEF CP Malik School of Life Sciences, Jaipur National University, Jaipur-302 025, India Email:
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LS – An International Journal of Life Sciences Vol. 1, Issue 2, August 2012 Contents 1.
Morpho-Anatomical Studies of Coccinia barteri (Hook. F.) Keay Cucurbitaceae EMI Roberts, GC Obute and BE Okoli
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2.
In vitro study of the effect of drought stress on the biochemical changes in Cicer arietinum L. Surya Nayar, P Sharma, V Soni and CP Malik
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3.
Combining Ability Analysis in Pearl millet [Pennisetum glaucum (L.) R Br.] KC Sharma, Ganga Ram and ML Jakhar
90
4.
Salt tolerance of Indian mustard: Physiological factors PC Sharma and TS Sinha
97
5.
Endophytic Fungal Metabolites and their potential: An Overview Kartikeya Tiwari and Payal Lodha
112
6.
Morphometrics and Morphology of Rice blue beetle Leptispa pygmaea (Baly.): (Coleoptera:Chrysomelidae) Tulsi Bhardwaj and KS Rana
118
7.
Comparative effect of different forms of tobacco smoking and their exposure rate on lung function in healthy smokers, non smokers and COPD patients Shivani Monga, Monika Tandon and CP Malik
121
8.
Molecular Strategies for Identification and Deployment of Gene(s) for Abiotic Stress Tolerance in Crop Plants Shabir H Wani, NB Singh, Jeberson SM, GS Sanghera, A Haribhushan, BU Chaudhury and MA Bhat
128
9.
Oxidative Stress and Anti-oxidant Machinery of Plants SK Thind and Prinka Goyal
143
10.
Patentability of Micro-Organism: Ethical or in Contravention to Nature Bhupendra Kumar and Ayush Yadav
159
11.
In vitro cloning of Pulicaria crispa: An important medicinal plant Jyoti Ushahra and CP Malik
171
LS - An International Journal of Life Sciences DOI : 10.5958/j.2319-118X.1.2.001
Morpho-Anatomical Studies of Coccinia barteri (Hook. F.) Keay Cucurbitaceae EMI Roberts1, GC Obute2 and BE Okoli*3 Ph.D Student, ² , ³Professor, Plant Biosystematics and Biotechnology Laboratory, Department of Plant Science and Biotechnology, University of Port Harcourt, Port Harcourt, Rivers state, Nigeria
1
1
Email id:
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ABSTRACT Detailed studies were carried out on the morphology and anatomy of Coccinia barteri (Hook. f.) Keay Cucurbitaceae in Nigeria, because the plant is medicinally useful and has not been fully described. Hand sections of vegetative parts were made and observed. Interesting and significant diagnostic features were observed and they include variations in the arrangement of vascular bundles in the midribs of the male and female collections. The morphological features of interest include the fruit size, shape and flower colour. The presence of a sclerenchymatous endodermis in the secondary structure is also diagnostic and is reported for the first time. The usefulness of these features in the taxonomic delimitation of this species is discussed. Keywords: Anatomy Coccinia barteri, morphology, taxonomy, vascular bundles
1. INTRODUCTION The genus Coccinia comprises 27 species that occur mainly in sub-Saharan Africa (Holstein and Renner, 2011); are eaten and used as vegetables. Coccinia barteri (Hook. f.) Keay is a herbaceous climber or trailer which is 15m long with a tuberous rootstock recorded as a wild plant in the lowland rainforests and swamp forests of some countries of West and East Africa (Flyman and Afolayan, 2006).The plant has several medicinal and social uses. A cold infusion of the plant is used in parts of Nigeria and West Cameroons for venereal diseases, taken internally; the powdered dried leaves are applied as a dressing for chancre. In Cameroons, the juice of the boiled leaves, mixed with a few drops of water is an ear-instillation for earache and deafness (Sandberg et al., 2005). It is also used to treat prostatic ailments (Noumi, 2010). The Akyem of Ghana take the contents of its fruit mixed with fresh lime juice and apply it with a feather to the umbilical cord, tied near the body with a strand of pineapple or plantain fibre of a newborn baby. The application is repeated till the cord falls off (Burkill, 1995). Despite the immense economic potentials of this species, Volume 1, Number 2, March-August, 2012
information on Coccinia barteri is meagre. Apart from the outline description of morphological features provided by Keay (1954), Jeffrey (1967) and Burkill (1995), which were based on herbarium materials, there are no reports of the investigation of its anatomy. This paper describes the interesting anatomical features occurring in Coccinia barteri; it aims at providing new and additional evidence for the delimitation of this species. 2. MATERIALS AND METHODS The material examined was obtained from living plants growing wild at different locations in Port Harcourt, Rivers State. Its taxonomic identity was confirmed using the Flora of West Tropical Africa (Keay, 1954) and the Flora of East Tropical Africa (Jeffrey, 1967). Fresh species specimens were used for the anatomical studies. Mature leaves and stems were collected from the middle portion of the plant. Hand sections were made following the method of Okoli (1987, 1992). Good sections were collected using a camel hair brush, stained with 1% Safranin for 2 min and rinsed in distilled water to wash off excess stain. The sections were then mounted 79
EMI Roberts, GC Obute and BE Okoli
temporarily in glycerine on microscopic slides and viewed under the microscope. Photographs were taken from good preparations with the aid of a Leitz Diaplan light microscope fitted with a Leica MPS camera; and a hand-held SONY (DSC-S950) digital camera.
plant shows a vasculature of four vascular bundles, three arranged in a triangular form and one being prominent at the abaxial groove. The vascular bundles are amphiphloic. The adaxial surface is slightly convex while the abaxial is sharply convex (Plates VI–VIII).
3. RESULTS
4.2 Petiole Anatomy
Table 1 shows the vegetative and floral morphological features of Coccinia barteri. Leaves are simple, glossy, coriaceous, puberulous, unlobed and 3–5 angled, or shallowly or palmately 3–5 lobed. The lobes are triangular, exstipulate and petiolate. Tendrils are bifid and coiled, arising at the nodes (at 90o) of each leaf petiole. Probracts are small and fleshy. Extra-floral nectaries are present. Flowers are unisexual, actinomorphic, puberulous, and conspicuous (yellow in colour) with a racemose inflorescence. Aestivation is induplicate-valvate. Corolla lobes are united to the middle, gamopetalous and campanulate. Calyx is gamosepalous. Stamens are three and synandrous. Filaments are united into a column; anther-lobes are variously folded (sinuous). Ovary is ellipsoid, sub-glabrous, unilocular, multicarpellary, and inferior with a parietal placentation. The style bears three stigmas, are free, thick and recurved. Staminodes are present. The fruit is a berry known as pepo, usually in racemose clusters; ellipsoid, rounded, smooth, green, when unripe and greenish–yellow when ripe. Seeds are ovate, compressed and have no endosperm. The plant is dioecious and parthenocarpic in nature, so pollination and fertilisation are limited (Plates I–V)
The distribution of the tissues in the petiole studied is shown in Plate IX. Common features include a layer of epidermal cells, which is followed by 4 tiers of collenchyma cells. There are 10–15 amphiphloic vascular bundles arranged ring-like in the parenchymatous ground tissue, with crescentric caps of sclerenchyma over the external phloem of each vascular bundle. 4.3 Stem Anatomy The stem has a circular outline. The primary stem anatomy shows a layer of epidermis, which is followed by cortical tissues that form a ring. This is followed by a strip of endodermis and a band of sclerenchyma cells. The vascular bundles (in a row) are surrounded by parenchyma cells (Plate X). The secondary structure stem has 5-tier collenchyma cells that follow the epidermis, which is a layer of cells. This is followed by sclerenchyma cells of about 4–5 layers, a sclerenchymatous endodermis and a ground tissue of parenchyma cells that surround the amphiphloic stele (Plates XI–XIII). 5. DISCUSSION
4. ANATOMY 4.1 Leaf Anatomy (Transverse Section of Lamina) The leaf is dorsiventrally flattened in outline with the mid-rib and major veins are prominent abaxially. The upper and lower epidermises are uniseriate. The palisade mesophyll consists of a layer of elongated cylindrical cells found close to the upper surface; the spongy mesophyll is confined to the centre of the lamina composed of 8–10 layers of cells, which are irregular in shape. The lamina is characterised by sclerenchymatous cells. The mid-rib is conspicuous and has about 4 layers of collenchyma cells following the epidermis. The midrib of the female plant has three vascular bundles arranged in a row, with two prominent bundles while the male 80
Morphology and vegetative anatomy are taxonomically valuable as they are used to delimit species from their relatives (Esau, 1977; Okoli, 1987; Fahn, 1990; Agbagwa and Ndukwu, 2004). They were therefore recorded in this study. Leaf morphology (architecture) is an essential tool in field and herbarium taxonomy, especially in the Cucurbitaceae (Jeffrey, 1964, 1980; Gill, 1988). Although, the leaf shape and size vary in C. barteri, its texture is coriaceous and is found to be diagnostic as it differs from other cucurbits. Though all the fruits of Cucurbitaceae are typically pepo, the shape, colour, and the surface texture of the fruit are diagnostic and useful in taxonomic studies. The size, shape and surface of the fruit of C. barteri are different LS - An International Journal of Life Sciences
Morpho-Anatomical Studies of Coccinia barteri (Hook. F.) Keay Cucurbitaceae Table 1: Summary of Morphological Features and Variations ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Characters Morphological variation Remarks ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1 Duration and Habit Perennial herbaceous climber or trailer Moderately hard 2 Stem Roundish, sub-glabrous Roots at nodes 3 Leaf type Simple, unlobed or palmately lobed Usually profuse 4 Leaf surface Coriaceous with prominent venation. Leaf dimensions i. Length (cm) Range Male: 19–23 Female: 15–23 Mean Male: 21.1 Female: 19.8 ii. Breadth (cm) Range Male: 18–22 Female: 14–23 Mean Male: 20 Female: 18.6 5 Petiole Length (cm) Range Male: 3–4 Female: 2–3 Mean Male: 3.8 Female: 2.5 6 Internode Length (cm) Range Male: 12–16 Female: 8–12 Mean Male: 14.2 Female: 9.6 7 Phyllotaxy Alternate 8 Inflorescence Raceme Dioecious 9 Flower Unisexual, actinomorphic, puberulous, and conspicuous Yellow in colour. 10 Calyx Fused Adnate to ovary in the female flower 11 Corolla Fused Adnate to calyx 12 Stamen 3 in number and synandrous. Anthers lobes are variously folded (sinuous) 13 Pistil 3 forked stigmas 14 Ovary Inferior, unilocular but multicarpellary with a parietal placentation. Placentas are 3 in number 15 Fruit Berry (pepo), many-seeded 1–5 on the stalk Shape Ellipsoid Colour Green, when unripe and yellowish green when ripe. Speckled with light yellow patches. Texture Smooth 16 Aestivation Induplicate-valvate 17 Seeds Small, compressed and ovate in outline, dull white in colour (dry). Has no endosperm -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
from other species at the genus and family levels, also noteworthy is the attachment of fruits (usually in racemose clusters) to the peduncle. These provide ease in the identification of the taxon. The leaf lamina of C. barteri has a thin epidermis (1-cell thick), closely packed palisade mesophyll (a layer) and densely packed spongy mesophyll, which are common features of mesophytic plants. Volume 1, Number 2, March-August, 2012
The vascular bundles of the leaf, petiole and stem of Coccinia barteri are similar to the type found in all the members of the Cucurbitaceae family, that is, amphiphloic vascular bundles (Cutler, 1978; Eames, 1961). Another significant diagnostic character is the vasculature of the mid-rib of the male and female species of the taxon. The female has three amphiphloic vascular bundles that are in a row with two being prominent; while the male has four vascular bundles with the less 81
EMI Roberts, GC Obute and BE Okoli
Plates (i) and (ii) Leaf; (iii) Male flower; (iv) Female flower and inflorescence of C. barteri, and (v) Fruits of C. barteri in racemose cluster; (vi) T/S of Lamina (Note one-layered palisade mesophyll); (vii) T/S of mid-rib of male C. barteri; (viii) T/S of mid-rib of female C. barteri; (ix) Petiole Anatomy of C. barteri (Note: bicollateral vascular bundles); (x) T/S of Primary Stem; (xi) T/S of Secondary Stem (Note bicollateral vascular bundles and well-developed sclerenchyma and xylem vessels); xii: Secondary Stem Anatomy showing tissue arrangement ep: epidermis, co: collenchymas, sc: sclerenchyma pa: parenchyma and en: endodermis; (xiii) Amphiphloic vascular bundle of C. barteri (xv): xylem and ph: phloem).
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Morpho-Anatomical Studies of Coccinia barteri (Hook. F.) Keay Cucurbitaceae
prominent three (3) in a somewhat ring-like form and the most prominent at the abaxial groove. This provides additional information for maintaining its distinction and agrees with the taxonomic values of the vascular bundle as earlier highlighted by Metcalfe and Chalk (1979). ACKNOWLEDGEMENTS The financial assistance provided under the World Bank Step-B Innovators of Tomorrow Grant to enable the authors purchase reagents and carry out field trips, is acknowledged with thanks. REFERENCES [1]
Agbagwa IO and Ndukwu BC (2004). The Value of Morphoanatomical Features in the Systematics of Cucurbita L. (Cucurbitaceae) species in Nigeria. African J. of Biotech, 3: 541-546.
[2]
Burkill HM (1995). The Useful Plants of West Tropical Africa 2 nd edn. vol.1 Royal Botanical Garden Kew. Cambridge University Press, Cambridge.
[3]
Cutler DF (1978). Applied Plant Anatomy. Longman Group Limited, London.
[4]
Eames AJ (1961). Morphology of the Angiosperms. McGrawHill Book Company, New York.
[5]
Esau K (1977). Anatomy of Seeds Plants. John Wiley Publisher, New York.
[6]
Fahn A (1990). Plant Anatomy 4 th edn. Perganon Press Plc. Headington Hill Hall, Oxford.
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[7]
FlymanFlyma MV and Afolayan AJ (2006). A Survey of Plants used as Wild Vegetables in 4 Districts of Botswana. Ecol. of Food and Nut., 45: 405-415
[8]
Gill ZO (1988). Taxonomy of Flowering Plants. AfricanaFEP. Publishers Limited, Onitsha. pp 338.
[9]
Holstein N and Renner SS (2011). A dated phylogeny and collection records reveal repeated biome shifts in the African genus Coccinia (Cucurbitaceae). BMC Evol. Biol.,11: 28.
[10] Jeffrey C (1967). Cucurbitaceae. In: Flora of Tropical East Africa, edited by Milne Redhead E and Polhill RM, Crown Agents, London. [11] Jeffrey C (1964). Key to the Cucurbitaceae of West Tropical Africa with a guide to localities of rare and little-known species. J. West African Sci. Assoc., 9: 79-97. [12] Jeffrey C (1980). A review of the Cucurbitaceae. Botanical J. Linnaean Soc., 81: 233-234. [13] Keay RWJ (1954). Flora of West Tropical Africa 2nd edn. Vol. 1. Crown Agents, London. [14] Metcalfe CR and Chalk L (1979). Anatomy of the Dicotyledons. 2nd edn Vol. 1. Clarendon Press, Oxford. [15] Noumi E (2010). Ethno medicines used for treatment of prostatic disease in Foumban, Cameroon. African Journal of Pharmacy and Pharmacology, 4: 793-805. [16] Okoli BE (1987). Morphological and Anatomical Studies in Telfairia Hooker (Cucurbitaceae). Fed. Repert., 98: 505-508. [17] Okoli BE (1992). Field, Herbarium and Laboratory Techniques. Mbeyi and Associates (Nig.) Ltd. Port Harcourt. [18] Sandberg F, Perera-Ivarsson P and El-Seedi HR (2005). A Swedish Collection of Medicinal Plants from Cameroon. J. of Ethnopharm, 102: 336-343.
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LS - An International Journal of Life Sciences DOI : 10.5958/j.2319-118X.1.2.002
In vitro study of the effect of drought stress on the biochemical changes in Cicer arietinum L. Surya Nayar1, P Sharma², V Soni³ and CP Malik4* ² ³School of Life Sciences, JNU, Jagatpura, Jaipur (302025), India Advisor, School of Life Sciences, Jaipur National University, Jaipur, Rajasthan, India
1, , 4
*Email id:
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ABSTRACT Addition of PEG 6000 in the growth medium decreased the germination and growth of Chickpea (Cicer arietinum L. cv. C-235 and BG-391) seedlings. Seedlings were subjected to MS medium as control, and 1, 2, 3, 4 and 5% PEG for 30 days. Evaluated traits in this study were invertase, β-amylase, acid phosphomonoesterase and protein. The results showed that the PEG treatments reduced the activity of invertase, β-amylase, acid phosphomonoesterase and protein. Final results indicate that C-235 cultivar is tolerant than BG-391 towards the drought stress. Keywords: Acid phosphomonoesterase, β-amylase, chickpea (Cicer arietinum L.), drought stress, invertase, protein Abbreviations: INVER: invertase, PEG: polyethylene glycol, SAT: Semi Arid Tropics
1. INTRODUCTION Environmental stresses, such as drought, salinity, cold and heat cause adverse effects on the growth of plants and the productivity of crops. Abiotic stress is the primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50%. Abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity (Wang et al., 2001). Water stress in its broadest sense encompasses both drought and salt stress. Drought and salinity are becoming particularly widespread in many regions, and may cause serious salinissation of more than 50% of all arable lands by the year 2050 (Bray et al., 2000). Drought and salt stress, together with low temperature, are the major problems for agriculture because these adverse environmental factors prevent plants from realising their full genetic potential. Chickpea (Cicer arietinum L.) is an important cash crop and cool-season food legume (pulse) with greater 84
nutritional value, accounts for higher consumption and economic importance of Rajasthan and all over the world. Chickpea is an important crop of Semi Arid Tropics (SAT) and the West Asia and North Africa. Closure of stomata may result from direct evaporation of water from the guard cells with no metabolic involvement. This process of stomatal closure is referred to as hydropassive closure. This process seems to be ABA regulated. Under the water deficit condition the pH of xylem sap increases, therefore, promoting the loading of ABA into the root xylem and its transport to the shoot (Hartung et al., 2002). Environmental conditions that increase the rate of transpiration also result in an increase in the pH of leaf sap, which can promote ABA accumulation and lead to reduction in stomatal conductance (Wilkinson and Davies, 2002; Davies, 2002). β-amylase expressed during stress has been shown to play a major role in transitory breakdown of starch (Scheidig et al., 2002). Plant invertases are essential for growth processes since they make sugars available for LS - An International Journal of Life Sciences
In vitro study of the effect of drought stress on the biochemical changes in Cicer arietinum L.
cell elongation. In maize, activity of vacuolar and cell wall-bound acid invertases predominates during ovary and early kernel development, whereas Sucrose synthase also becomes important during the storage phase of grain-fill (Zinselmeier et al., 1995; Weschke et al., 2000). Drought stress decreases activities of both vacuolar and cell wall-bound acid invertase during kernel development (Zinselmeier et al., 1995), with parallel reductions in ovary growth and concentration of hexoses. Soluble invertase expression is an early target of drought stress during the critical, abortion-sensitive phase of young ovary development in maize (Andersen et al., 2002). Acid phosphohomonoesterase activity is known to contribute to resistance under salt and water stress by maintaining a certain level of inorganic phosphate (Olmos and Hellin, 1997). Although much supporting evidences on the role of nonantioxidative enzymes under drought are available, there are no specific information pertinent to Chickpea. The objective of the present study was to evaluate the effect of drought stress on non-antioxidative enzymes in Chickpea, and also to select the best variety among two against drought stress under in vitro conditions. 2. MATERIALS AND METHODS 2.1 Plant Material and Drought Stress The seeds of C. arietinum cv. BG-391 and C-235 were collected from Agriculture Research Station, Durgapura, Jaipur. Seeds were surface sterlised before transferring on the culture medium and for this purpose the seeds were washed in running tap water and transferred to 0.1% (w/v) HgCl2 solution for 3-4 min and finally rinsed three to four times with sterile distilled water. The seeds were inoculated on nutrient agar medium under aseptic conditions by using laminar airflow cabinet pre-sterlised by ultraviolet radiation. In this experiment different concentrations of polyethylene glycol 6000 (PEG) were added to the nutrient medium. The sterilised seeds of both of the genotypes of chickpea were cultured on plain hormone free MS Murashige and Skoog (1962) medium as control and others were supplemented with different concentrations of PEG (0, 1, 2, 3, 4 and 5%). All the cultures were incubated in the growth room having Volume 1, Number 2, March-August, 2012
the light intensity of 1200 lux from fluorescent tubes and incandes-cent bulbs at a temperature of 26±20C. A photo- period of 16 h was maintained with the help of a timer. After 30 days, the leaves of developed plantlets were used for a biochemical assay in order to determine the effect of PEG-induced- drought stress on various biochemical parameters of C. arietinum cv. BG-391 and C-235. 2.2 Extraction Enzymes The fresh plant material (0.5 g) was homogenised in 0.1 M chilled/ice cold phosphate buffer, pH 7.0 and centrifuged at 10,000 rpm for 10 min at 4ºC and the supernatant was treated as an enzyme extract. The supernatant was used as a source of enzymes. 2.3 Assay of Enzymes β-Amylase (E.-C.- No. 3.2.1.2) activity was assayed according to the method Bernfeld’s method (1955). Enzyme activity was expressed in terms of mg maltose liberated per hour per mg protein. Acid invertase (E.-C.- No. 3.2.1.26) activity was determined by a modified method of Harris and Jeffcoat (1974). The procedure involved the breakdown of sucrose into glucose and fructose and estimating the amount of glucose (Sumnner, 1935). One unit activity was determined as the number of mg/ml of glucose liberated per hour per mg protein at 560 nm. The activity of acid phosphomonoesterase (E.-C.- No. 3.1.3.2) was assayed by using p- nitrophenyl phosphate as substrate (Zink and Veliky, 1979). Each unit of activity is defined as the mM p- nitrophenyl released per min per mg protein. Soluble protein content was determined according to Bradford’s method (1976) with BSA as the standard. 3. STATISTICAL ANALYSIS All data are expressed as means of triplicate experiments unless mentioned otherwise. Comparisons of means were performed using SPSS 9.0 software. Data were subjected to a one-way analysis of variance (ANOVA), and the mean differences were compared by lowest standard deviations (LSD) test. Comparisons with P
