JNKVV RESEARCH JOURNAL

Volume 44 Number 1 January - June 2010

Contents

Research Paper Resin production system in Guggul [ wightii (Arnott) Bhandari] 1 Moni Thomas, Atul Shrivastava and S.S. Tomar

Upland rice in Madhya Pradesh: Problems and Prospective 9 I.M. Khan, R.K. Tiwari and S.K. Rao

Production, purification and characterization of phytase from Bacillus subtilis 19 Namrata Verma, Keerti Tantwai, L.P.S. Rajput, Sunil Kumar, Iti Gontia and Sharad Tiwari

Production of phytase from soil isolates ofPseudomonas sp. using agricultural 25 byproduct substrates Yuvraj Soni, Keerti Tantwai, L.P.S. Rajput, Sunil Kumar, Iti Gontia and Sharad Tiwari

Analgesic activity of aqueous and alcoholic extracts of Noni (Morinda citrifolia ) 31 by hot plate method Bharti Jethani, R.K.Sharma and Sudhir Kumar Jain

Detection of infectious bronchitis virus in suspected post-mortem field tissue 35 samples by ELISA Sudhir K. Jain, Hemlata Jain, Megha Kadam Bedekar, Bikas Chandra Sarkhel and Sanjeev Singh

Integrated nutrient management in rice wheat cropping system 39 B.M. Maurya, Jyoti Dekate and V.B. Upadhyay

Evaluation of site specific nutrient management on productivity and economics 45 of rice-wheat crop sequence V.B. Upadhyay, S.K. Vishwakarma and Vikas Jain

Effect of inorganic, organic and sources of nitrogen on growth and yield of 49 fodder oat (Avena sativa L.) Anay Rawat and S.B. Agrawal

Response of sorghum [Sorghum bicolor (L.) Moench] cultivars to nitrogen and phosphate 53 solubilizing bacteria under different cutting intervals B.D. Ghode, Seema Bhadauria and S.D. Sawarkar

Jawaharlal Nehru Krishi Vishwavidyalaya Jabalpur 482004 (Madhya Pradesh) JNKVV RESEARCH JOURNAL ISSN : 0021-3721 Registration No.: 13-37-67

Effects of inclusion of soybean and potato on nutritional and sensory quality of 57 rice based extrudates Rajendra Singh Thakur and A.K. Tomar

Extracellular enzyme profile of oyster mushrooms as affected by gamma radiation 63 Alpana Singh and Anshu Verma

Screening of pesticides againstSclerotium rolfsi i causing collar rot of soybean 67 Ashish Kale and R.K. Varma

Microcontroller based seed germination testing device for soybean 75 A.K. Rai and Bharati Dass

Web enabled software for integrated farm production management of 79 major crops grown in central India for sustainable growth Bharati Dass and A.K. Rai

Molecular characterization of BoLA-DRB3.2 allele in Nimari breed of cattle by 83 single strand conformational polymorphism (SSCP) R. Ranjan, C.D. Bhong, K.N. Chavan, S.N.S. Parmar and C.G. Joshi

Information seeking behaviour of extension personnel regarding improved 87 agricultural technology N.K. Khare, A.K. Wankhade and Tapan Chandra Kar

ICT based Kisan Mobile Sandesh-an innovative approach 93 Nalin Khare and Abhay Wankhade

Hypsometric analysis of Kanhiya Nala watershed using 97 geographical information system S.K. Sharma, S. Tignath and U.C. Chaube

Effect of modified atmosphere storage (under vacuum and nitrogen) 101 on shelf life of rice bran S.P. Srivastava and A.L. Basediya

Effect of process and operational parameters of extrusion cooking of sorghum, horse 107 gram and defatted soy flour blends to produce protein rich ready-to-eat food snacks A.L. Basediya, S.P. Shrivastava and Sheela Pandey

An approach for possibilities for e-preservation of library holdings 113 Siddarth Nayak, Sharad K. Jain and P.S. Nayak

Published by : Dr. S.S. Tomar, Dean, Faculty ofAgriculture, JNKVV, Jabalpur 482 004 (M.P.),India Printed at : M/s Fortune Graphics, Sahu Mohalla, Near Kochhal House, Jabalpur 482 002 (M.P.) STATEMENT OF OWNERSHIP FORM IV (See Rule 8)

Place of Publication : Jabalpur (Madhya Pradesh), India

Periodicity of Publication : Biannual

Publisher's Name : Dr. S.S. Tomar Indian Dean, Faculty of Agriculture JNKVV, Jabalpur 482 004 (M.P.), India

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Editor's Name : Dr. Mohan S. Bhale Indian Senior Scientist Department of Plant Pathology JNKVV, Jabalpur 482 004 (M.P.), India

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Dated the 31st December, 2011 Sd/- S.S. Tomar Publisher A Publication of Jawaharlal Nehru Krishi Vishwavidyalaya Jabalpur 482 004 (Madhya Pradesh) India Phone : PBX : (+91) (0761) 2681074, 2681200 Fax : (+91) (0761) 2681074, 2681200 Website: www.jnkvv.nic.in

JNKVV Research Journal Editorial Board

Patron Prof. Gautam Kalloo Vice Chancellor, JNKVV, Jabalpur Chairman Dr. S.S. Tomar Dean, Faculty of Agriculture & Director Research Services, Jabalpur Members Dr. O.P. Veda Director Instruction, Jabalpur Dr. P.K. Bisen Director Extension Services, Jabalpur Dr. D.K. Mishra Dean, College of Agriculture, Jabalpur Dr. T.K. Bhattacharya Dean, College of Agricultural Engineering, Jabalpur Editor Mohan S. Bhale Co-Editor Abhishek Shukla

General Information: JNKVV Research Journal is the publication of J.N. Agricultural University (JNKVV), Jabalpur for records of original research in basic and applied fields of Agriculture, Agricultural Engineering, Veterinary Science and Animal Husbandry. It is published twice a year. The journal is abstracted in CAB International abstracting system, Biological Abstracts, Indian Science Abstracts. Membership is open to all individuals and organizations coping with the mission of the University and interested in enhancing productivity, profitability and sustainability of agricultural production systems and quality of rural life through education, research and extension activities in the field of agriculture and allied sciences.

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ISSN : 0021-3721 Registration No. : 13-37-67

Published by : Dr. S.S. Tomar, Dean, Faculty of Agriculture, JNKVV, Jabalpur 482 004 (M.P.), India Printed at : M/s Fortune Graphics & Scanning Centre, Sahu Mohalla, Golebazar, Jabalpur (M.P.) JNKVV Res J 44(1): 1-7 (2010)

Resin production system in Guggul plant [Commiphora wightii (Arnott) Bhandari]

Moni Thomas, Atul Shrivastava and S.S. Tomar Directorate of Research Services Jawarharlal Nehru Krishi Vishwa Vidyalaya Jabalpur 482 004 (MP)

Commiphora spp. () are native East (two isomers E and Z) is an African species found in Kenya, Uganda, Tanzania, effective anti-hyperlipidemic agent obtained from the gum Ethopia, Somalia (Beentje 1994; Gillet 1991) India, resin of guggul , C. wightii (Dev 2006; Wang et al. Aghanisthan, Arabia and Baluchistan in Pakistan 2004; Satayavati et al. 1969; Tripathi et al. 1968; Singh (Varier1994; Atal et al.1975). Commiphora wightii L. (Syn. et al.1994). Over exploitation and slow growth of the tree Commiphora mukul Engl, Balsamodendron wightii Arn., which is associated with poor seed set, make this plant B. mukul Hook. ex stocks) belongs to the family an endangered species (Kumar et al. 2004).The Burseraceae which is a large pantropical family, forming biotechnogical production of guggulsterone has received an important element of the flora of both rain forests and attention as a promising alternative source.The plant is arid areas (Bhandari 1995). C wightii plant is known as distributed in western India, the (Kumar and Indian is distributed in the arid, rocky tracts of Shankar 1982). , , Karnataka, Rajputana, Bellari, Baluchistan, Assam, Berar and Mysore states of India, Earlier the plant was found in abundant in and Sindh and Baluchistan states of Pakistan (Varier Rajasthan, Gujarat and Madhya Pradesh but currently 1994; Atal 1975) . In Gujarat, this species is mainly found threatened because of significant declines in population in Kachchh and in some parts of Saurashtra regions (IUCN2004) and faulty extraction methods employed by (Sabnis and Rao 1983; Shah 1978). In Madhya Pradesh, traditional guggul resin collectors (Thomas et al. 2009). It it is found in Bhind, Morena, Shivpuri, Sheopur and Gwailor came in vulnerable list in the 1997 IUCN Red list as districts (Thomas et al. 2009,b). vulnerable (VU) and in 2004 data deficiency (DD) (IUCN 2004). The Government of India recently banned export Members of Burseraceae family are the source of the Guggul gum (IUCN 2004), due to its high market of well known and franchincense of the Bible (Smith price in international trade. et al. 1997). Resins from the members of the family Burseraceae play an integral role in the national, regional C. wightii grows to 1.0-2.5m in height and and local economies in Ethiopia, Sudan, India and have sharp spines and papery bark (Kumar and Shankar Somalia (Leminih et al. 2003; Farah 1994). Resins from 1982). have aromatic smell. The guggul gum, many woody species from African and Asian countries extracted from the stem of C. wightii. The plant is known are used commercially in the pharmaceutical, food, for its medicinal and pharmacological properties (Thomas cosmetics and industries (Coates Palgrave 2002; et al. 2010 a). It controls rheumatoid arthritis, obesity Leminih et al. 2003). The term resin and gum are often and peptic ulcer (Atal et al. 1975). Hypocholestermic, used to describe any stick plant exudates which could hypolipdimic, anti-inflammatory,anti-rheumatic and anti- include a range of substances. Langenheim (2003) defines fertility activities (Nityanand and Kapoor 1971 a,b; Kapoor resin as a 'lipid soluble mixture of volatile and non volatile et al. 1979; Kakrani 1981 a,b; Tajuddin et al.1994; Singh terepenoid and or phenolic secondary compounds that et al. 2001) also have been reported. are i. usually secreted in specialized structures either internally or on the surface of the plant and ii. of potential Resin ducts signifinance in ecological interactions. As such resins play no role in the primary or fundamental physiology of the plant. The main components of gums are water soluble The oleo-gum resin of C. wightii is generally known as whereas resins are lipid soluble. Guggul is of wide medicinal and commercial value

1 (Hocking 1993; Kirtikar and Basu 1935). One of the the radial walls gets undulated. The cytoplasm grows distinguishing features of Burseraceae family is the dense with mitochondria, endoplasmic reticulum and resin presence of resin ducts in the bark. Guggul is secreted droplets in the injured epithelial cell and its neighbouring form the bark as a result of wound or incision on the bark cell in comparison to that in the normal cell. In the injured (Krishnamurthy and Shiva 1977). The associated cell there is a rapid conversion of lipid material into resin anatomical characteristics of the bark of C. wightii (Setia droplets. Among the resin droplets, dense vesicles remain et al. 1977; Nair et al. 1981; Shah et al. 1982) as well as scattered suggests the transport of the polysaccharide the ultrastructural details of secretion, seasons of (resin) material to the plasmalemma of the inner tangential production and methods of enhancing the yield have been wall of the epithelial cell in the injured tissue. Apoplastic reported (Bhat et al. 1989). transport of the resin occurs as it passes through the loose matrix of the inner tangential wall of the epithelial cell and gets accumulated in the resin duct lumen. Structure of Resin duct Distortion of mitochondria and plastids, disintegration of the plasmodesmatal connections and appearance of Guggul is synthesized in specialized cells and collected autophagic vacuoles after 45 minutes of an injury is the in the lumen of the resin duct (Setia 1976; Setia et indication of the beginning of autolysis of the epithelial al.1977; Nair et al. 1981b). The 'resin canals' or 'resin cell and the product of it is gum-resin. Senescence and ducts' are found in the phloem of larger veins of the death of the neighbouring cells increases the exudation and in the soft base of the stem. The resin ducts are (Shah et al. 1982). bordered by a layer of dense epithelium in the secondary phloem. The epithelial cells have an organelle rich In an injured epithelial cell an increase of lipid cytoplasm with abundant lipid and resin droplets. Due to droplets in the cytoplasm is presumed to a be the the presence of resinous material amidst the cellulose precursors of resin material (Nair et al. 1981b). An intense microfibrils, the inner tangential wall of these epithelial lipase activity in the epithelial cells of the injured tissue cells bulge to attain convex shape and gets thicker than may be helping in the metabolism of lipids into fatty acids the outer tangential cell wall, in the process of resin which is shunted into alternate pathways contributing to transportation into the duct lumen (Shah et al. 1982). the formation of resin fraction (Shah et al. 1980). Accelerated senescence and death of cells associated The resin ducts are not differentiated in globular with increased gummo-resinosis in the injured tissue and stage embryos but only at torpedo stage in somatic release of ethylene due to injury is an important event embryos in vitro. Yet the resin ducts found in vitro formed (Field 1981). Senescence and autolysis that follow can somatic embryos were quite distinct, well-differentiated, be the consequence of loss of compartmentalization, evenly distributed and comparable to those formed in vivo. release and dispersal of hydrolases in the cell, The epithelial cells lining the resin ducts and those disorganization and disruption of organelles (Shah et al. adjoining it are rich in cytoplasm with high metabolic 1982). activity. Such resin ducts are formed before differentiation of vascular bundles as evident from the transverse section The secretory ducts occur in association with of cotyledonary stage embryo. A large number of resin secondary phloem in the main stem. They are ducts were present in the hypocotyls region of 5-6 week discontinuous, oriented parallel to the longitudinal axis of old cotyledonary stage embryo. These ducts separated the stem and anastomose tangentially. April and May the cortical region from pith region. Resin ducts develop are the peak months for guggal tapping as established in all parts of the seedlings with branches even in the by localization of resin in the sectioned material using young cotyledons. bright field and epifluorescence microscopy (Bhatt et al.1989).

Mechanism of resin exudation in C. wightii Can the flow of guggul accelerated?

Upon injury to the plant, the gum-resin exudes immediately and flows down the trunk continuously for long period, Understanding the mechanism of flow of guggul from the simultaneous intensified morphological changes in the injured tissue,a thought that stuck upon was can widening epithelial cells 45 minutes after the injury (Nair et al. of the lumen of the resin duct lead ti increase in the flow 1981a). On injury of the bark,the epithelium of the resin of Guggul? There is a strength to this school of thought ducts collapses and the outer tangential wall of the because the development and widening of gums-resin epithelial cells thickens as the inner tangential wall, while canal in young stem occurs schizogenously (Setia et al.1977; Bhatt 1987). This lead to affort to experiment on

2 the widening of the lumen of the resin duct. Among the When properly carried out the tapping of wood exudates various plant growth regulators applied on the stem with does not necessarily have to reduce tree vitality, the ability lanolin paste,only kinetin increased the lumen size, while to grow and reproduce under the present environmental auxin and morphactin had adverse effect causing increase conditions. However, increasing demands for exudates in the number of epithelial cells (Setia and Shah 1979), and their commercial extraction often result in harvesting whereas ethephon enhanced 22 times oleo-gum techniques that maximize short-term economic gains with production (Bhatt et al. 1989). little consideration for the long-term ecological consequences (Coppen 1995 a; b). Resin production in plants Tapping Generally, resins are expensive to produce in terms of grams of glucose per grams of material (Gulmon and Commericially, C. wightii is tapped to extract Guggul. Mooney 1986). In Bosewelia papyrifera - a slow growing Oleo-resin is obtained from the living bark (i.e. phloem). resin yielding plant species occurring on infertile and dry Local tappers make incisions in the bark of the trunk soils (Ogbazghi 2001), assimilates are likely to be invested using various implements ranging from simple knife to more in storage of carbohydrates and resin synthesis even axe. Though the incision has to be made not beyond than in growth (Loomis 1932; Bryant et al. 1983; Herms the bark of the plant but practically all injury travels to the and Mattson 1992). Indeed, in the first 3-4 years young heart wood. Even the specific axe, locally known as Boswellia plants die back aboveground at the end of the a'Mingaf' and 'Mitchie Golledge' knife could help in limiting rainy period and produce tuber shaped tap roots to the tapping injury to the plant (Thomas et al. 2010 a). An overcome the long dry season (W Ogbazghi unpublished improved taping technique, using 'Mitchie Golledge' knife, data). Also, adult produce large tuber-like structures coupled with ethephon application devised enhanced the from the base of the trunk into the soil, presumably for guggul production by about 22times over that obtained storage use and soil anchoring. The annual extraction of by traditional practices (Bhatt et al.1989). The traditional frankincense depends on stored carbohydrates, leading tapping methods used for obtaining guggal, the oleo-resin in the long term to the depletion of carbohydrate reserves from C. wightii, are unproductive and destructive. that, under non-tapped conditions, would be used for vegetative growth and reproduction. Plants allocate Krishnamurthy and Shiva (1977) carried out a carbohydrates for tapping experiment on C. wightii with six points along the tree stem, the 'normal' intensity. The resin that exudes is (i) resource capture and growth left to solidify into small tears or lumps; collectors harvest (ii) reproduction them every 3 weeks and the cuts are re-opened to allow (iii) storage purposes and a steady flow of the resin. Each tree is revisited at least (iv) the protection of already captured resources. seven times per harvest season. Tapping is discontinued during the rainy period because yields are low and the Therefore tapping for resin is likely to affect the resin is washed away from the stem. pattern of carbohydrate allocation in a tree, as it enhances the competition for assimilates between growth processes Impact of tapping on plants and resin production.

Deterioration of growth processes as a result of commercial Why plants exudates? tapping has been studied extensively for plantation trees. For example, (Dijkman 1951) found that heavy tapping of Wood exudates are generally assumed to provide plant latex in Hevea trees during 5 years in south-east Asia defence, because they seal off injured tissue, prevent resulted in a decrease of about 50 per cent in the dry desiccation from injured tissue, protect against attack wood production of trees. Kärkkäinen (1981) found that by insects and fungi and prevent further injury through wood increment of Scots pine trees dropped by 35 per decay (Howes 1949; Hart 1989; Phillips and Croteau cent when the resin yield was doubled. Empirical field 1999). Excessive collection of resin and latex can be observations have revealed the negative effect of destructive as it may weaken the tree and causes overtapping for wood exudates, such as premature tree carbohydrates to be spent on exudates that might death and enhanced risk of insect and fungus infections otherwise have been allocated to growth and reproduction (Torquebiau 1984; Jamal and Huntsinger 1993). The (Dijkman 1951; Karkkainen 1981). impact at the individual and population levels of

3 over-exploitation of non-timber products other than wood from current photosynthesis, as the supply of stored exudates, such as leaves, fruits and bark, has been carbohydrates is not sufficient (Lieutier et al.1993). In B. demonstrated for many different naturally growing plant papyrifera, tapping is always undertaken in the leafless species (Peters 1996; Ticktin 2004). dry period, making the synthesis of frankincense dependent on stored carbohydrates. The same is true for The small trees would suffer more from the tapping C. wightii. treatments than large trees, as the former have fewer stored carbohydrates available. Tapping activities coincide Production of guggul in vitro with the sexual reproduction cycle of the tree (i.e. and seed production). Sexual reproduction decreased with C. wightii is a slow growing plant. The suitable age of increasing tapping regime irrespective of tree size. Overall, tapping the plant is after it is six years. The existing large trees tended to produce slightly heavier seeds than natural stand of the plant has reduced drastically due to small trees, and seeds from non-tapped trees were heavier destructive tapping. This resulted is placing the plant in than those from tapped trees. In the stands where tapping RET category in India. Reduction of the production of was prohibited changes in tapping regimes had the guggul in India is a reflection of the destructive tapping. greatest effect on sexual reproduction. Trees subjected to annual tapping always showed the lowest sexual In recent years the demand for guggul has reproduction. Non-tapped trees produced three times as increased in Indian pharmaceutical industry. Thus there many healthy and filled seeds as tapped trees. is an increasing pressure on the existing natural plant Germination success was highest in stands with non- stand. In order to cope this rising demand plant tapped trees (>80%) and lowest for those with tapped biotechnological methods are being pursued for rapid trees (<16%).Tapping for frankincense results in limited micro-propagation of C. wightii. Moreover, in vitro flower and fruit production, and low production of mainly technology will also be helpful in developing alternative non-viable seeds in B. papyrifera. We argue that tapping methods of guggulsterone production, thereby directly causes competition for carbohydrates between relieving pressure on the natural resources. Several frankincense production, and fruit and seed setting. attempts have been made in the last two decades to Consequently, the current tapping regimes will cause tree develop methods for its micro-propagation through in vitro exhaustion and eventually a decline in vitality. Tapping techniques like clonal propagation (Brave and Mehta 1993) may potentially reduce natural regeneration of the species and somatic embryogenesis through zygotic embryos (Toon et al. 2006). (Singh et al. 1997), but satisfactory results are yet to be obtained. Isolation of embryonic cultures and achievement Tapping for frankincense results in limited of somatic embryogenesis in callus cultures (Kumar and carbohydrate availability and therefore causes low Ramawat 2000) opens the possibilities of in vitro production of mainly non-viable seeds. Sunnichan et al. production of guggulsterones. (2005) suggested that observed low-fruiting levels during the leafless period in Boswellia serrata was the result of The high productive callus cultures of C. wightii, limited resources stored in the stem. Latta et al. (2000) from stock cultures maintained on MS (Murashige and found that frequent cutting or pruning of two agroforestry Skoog's) medium supplemented with 0.25mg/l 2,4,5- tree species resulted in a decrease of reserve non- trichlorophenoxyacetic acid and 0.1mg/l kinetin,in shake structural carbohydrates. They suggested that flasks and bioreactor yielded 80µmg guggulsterone per continuation of cutting for extended periods depletes a litre culture suspension(Dass and Ramawat 2009).It is tree of its carbohydrate reserves and will decrease well established that secondary metabolites contents are biomass production. Resin production after deliberately low in cell cultures as compared to callus cultures( wounding of the stem in conifer species showed that trees Ramawat and Mathur 2007). with large crowns had a higher level of resin production than those with small crowns (Schopmeyer and Larson An achievement of highest frequency of shoot 1955; Mason 1971; Ruel et al.1998). The positive formation of C. wightii trees in vitro through forced auxiliary correlation between resin yield and tree size is probably branching on MS (Murashige and Skoog's) medium a result of the greater overall carbon budget of larger sized supplemented with 17.8µM benzyladenine (BA) and trees (Sterck and Bongers 2001; Sterck et al. 2005), which 18.6µM kinetin, 100mg/litre glutamine,10mg/litre thiamine allows for higher allocation to exudate production. Whether hydrochloric acid and 0.3 per cent activated charcoal and conifer trees that are tapped use stored carbohydrates or its successful establishment in soil (Barve and Mehta current assimilates for resin production is unclear. It is 1993) have opened up possibility to propagate elite guggul suggested that in juvenile trees resin production originates producers from natural selection.

4 As evident from the histology of in vitro formed Coppen JJW (1995 a) Flavours and Fragrances of Plant somatic embryos, the resin ducts were not differentiated Origin. Non-Wood Forest Products 1 FAO in globular stage embryos or in other words, resin ducts Coppen JJW (1995 b). Gums, Resins and Latexes of Plant were differentiated only at torpedo stage embryos. No Origin. Non-Wood Forest Products 6 FAO such tissue differentiation was observed in unorganized Dass S, Ramawat KG (2009) Studies on somatic cell callus. The resin ducts found in vitro formed somatic variability in Commiphora wightii (Arnott.) Bhandari embryos were quite distinct, well-differentiated, evenly for guggulsterone production. Natural Product distributed and comparable to those formed in vivo. The Radiance 8(5):532 -536 epithelial cells lining the resin ducts and those adjoining Dev S (2006) A selection of prime Ayurvedic plants drugs(ed) Anamaya Pub New Delhi it are rich in cytoplasm and with high metabolic activity. Such resin ducts were formed before differentiation of Dijkman M J (1951) Hevea: Thirty Years of Research in the Far East. University of Miami Press Coral Gables vascular bundles as evident from the transverse section Florida of cotyledonary stage embryo, and were oriented parallel Farah AY (1994) The milk of the Boswellia forests: to the longitudinal axis. A large number of resin ducts frankincense production among the pastoral were present in the hypocotyls region of 5-6 week old Somali.EPOS Department of Social and Economic cotyledonary stage embryos. These ducts separated the Geography Uppasala University Uppasal Sweden cortical region from pith region. Resin ducts develop in all Field RJ (1981) The effect of temperature on ethylene parts of the seedlings with branches even in the young production by leaf tissue of Phaseolus vulgaris L. cotyledons (Kumar et al. 2004). Annal Bot 47:251-223 Gillet JB (1991) Burseraceae.In: Flora of Tropical East Africa.(A A Balkema,Rotterdam and Brookfield ed). Acknowledgement The authors are grateful to the ICAR, New Delhi and the Project Coordinator, Network Project on Gulmon SL, Mooney HA (1986) Costs of defence and their Harvesting,Processing and Value Addition of Natural Resins effects on plant productivity. On the Economy of Plant and Gums for financial assistance.The authors are also Form and Function (ed. Givnish TJ), pp 681-698 thankful to Prof Guatam Kalloo, Vice Chancellor, JNKVV Cambridge University Press London UK Jabalpur, for critical suggestions and inputs. Hart JH (1989) The role of wood exudates and extractives in protecting wood from decay. Natural Products of Woody Plant Vol 2 (ed. Rowe JW) pp 861-880 References Springer-Verlag Berlin Germany Herms DA, Mattson WJ (1992) The dilemma of plants: to Atal CK (1975) Source of guggul in Indian system of grow or defend. The Quarterly Rev Bio 67 283-335 medicine.Econ Bot 29:208-218 Hocking D (1993) Trees for Drylands. Oxford &IBH publishing Atal CK, Gupta OP, Afaq SH (1975) Commiphora mukul Co New Delhi source of gugal in Indian systems of medicine. Econ Howes FN (1949) Vegetable Gums and Resins. The Bot 29(3):209-218 Chronica Botanica Company Waltham MA Beentje HJ (1994) Kenya Trees,Shrubs and Lianas. The IUCN (2004) ICUN Red list of threatened species website National Museum of Kenya Nairobi Kenya Jamal A, Huntsinger L (1993) Deterioration of a sustainable Bhandari MM (1995) Flora of the Indian desert.3rd ed MPS agro-silvo-pastoral system in the Sudan: the gum REPROS Jodhpur gardens of Kordofan. Agroforestry Systems 23:23-38 Bhatt JR (1987). Development and structure of primary Kakrani HK (1981,a) Guggul-review.Indian Drugs 9:417-421 secretory ducts in the stem of Commiphora wightii Kakrani HK (1981,b) Flavonoids from the of (Burseraceae). Annal Bot 60:405-416 Commiphora mukul. Fitotewrapia 52(5):221-222 Bhat JR, Nair MNB, Mohan Ram HY (1989) Enhancement of Kapoor NK, Sukh Dev, Nityanand S (1979) Process for oleo-gum resin production in Commiphora wightii obtaining hypolipidemic and antiplatelet fractions by improving tapping technique. Curr Sci from guggul resin. Indian patent no.148265 dt 58:346-349 6.4.1979 Brave DM, Mehta AR (1993) Clonal propagation of mature Kärkkäinen M (1981) Increasing resin content in pine and elite trees of Commiphora wightii. Plant Cell Tissue spruce stem wood for higher by-product yield. Organ Cult 35:237-244 Communicationes Instituti Forestalis Fenniae Bryant JP, Chapin FSIII, Klein DR (1983) Carbon/nutrient 96:1-81 balance of boreal plants in relation to vertebrate Kirtikar KR, Basu BD (1935) Indian medicinal Plants Vol 1 herbivory. Oikos 40 357-368 Bishen Singh Mahendra Pal Singh Dehra Dun Coates Palgrave K (2002) Trees of Southern Africa.Struik Krishnamurthy T, Shiva MP (1977) Salai guggul from Publishers Cape Town South Africa Boswellia serrata Roxb its exploitation and utilization. Indian Forester 103:466-474

5 Kumar S, Shanker V (1982) Medicinal plants of the Indian Phillips MA, Croteau RB (1999) Resin-based defenses in deserts:Commiphora wightii (Arnott) Bhandari. J conifers. Trends in Plant Sci 4:184-190 Arid Environment 5:1-11 Ramawat KG, Mathur M (2007) Factors affecting production Kumar S, Ramawat KG (2000) Somatic embryogenesis in of secondary metabolites, In. Biotechnology: callus and cell cultures of Commiphora wightii: Secondary metabolites. Ramawat K G and Merillon Problems, perseverance and prospects. Proc Nat J M (eds)Science Publishers Inc Enfield USA pp Symp Potential of Plant Biotechnology in India at 59-101 Jay Narayan Vyas University Jodhpur 12 Ruel JJ, Ayres MP, Lorio PLJr (1998) Loblolly pine responds Kumar S, Sonie KC, Ramawati KG (2004) Development of to mechanical wounding with increased resin flow. resin canals during somatic embryogenesis in Canadian J Forest Res 28: 596-602 callus cultures of Commiphora wightii. Indian J Sabnis SD, Rao KSS (1983) Rare and endangered endemics Biotech 3:276-270 of assessment of threatened plants of India. Proc Langenheim JH (2003) Plant resins-Chemistry, Evolution, Seminar Dehradun pp 14-17 Ecology and Ethnobotany. Timber Press Portland Satyavati GV, Dwarkanath C, Tripathi SN (1969) Experimental and Cambridge studies on the hypercholesterolemic effects of Latt CR, Nair PKR, Kang BT (2000) Interactions among cutting Commiphora mukul Engl (Guggul). Indian J Med frequency, reserve carbohydrates, and postcutting Res 57:1950-62 biomass production in Gliricidia sepium and Schopmeyer CS, Larson PR (1955) Effects of diameter, crown Leucaena leucocephala. Agroforestry Syst 50:27-46 ration, and growth rate on gum yields of slash and Lemenih M, Abebe T, Olsson M (2003) Gum and resin long leaf pine. J Forest 53: 822-826. resources from some Acacia, Bosewellia and Setia RC (1976)Morphogenetic studies in gum producing Commiphora species and their economic plant cells. Ph.D. Thesis Sadar Patel University contributions in Liban South-east Ethoipa. J Arid Vallabh Vidyanagar India Envir 55:465-482 Setia RC, Shah JJ (1979) Histological,histochemical and Lieutier F, Garcia J, Romary P, Yart A, Jactel H, Sauvard D ultrstructure aspects of gum and gum-resin (1993) Inter-tree variability in the induced defense producing structures in plants. In Ann Rev Plant Sci reaction of Scots pine to single inoculations by (ed.) Malik C P Kalyani Publi New Delhi 315-332 Ophiostoma brunneociliatum, a bark-beetle- Setia RC, Parthasarathy MV, Shah JJ (1977) Development, associated fungus. Forest Eco and Manag histo-chemistry and ultrastructure of gum-resin 59:257-270 ducts in Commiphora mukul Engl. Annals Bot Loomis WE (1932) Growth-differentiation balance vs. 41:999-1004 carbohydrate-nitrogen ratio. Proc Ameri Soc Horti Shah GL (1978) Flora of Gujarat state.University Press Sadar Sci 29:240-245 Patel University Vallabh Vidhyanagar Anand Mason RR (1971) Soil moisture and stand density affect Shah JJ, Subramanyam SV, Nair MG, Patel, KR (1980) oleoresin exudation flow in a loblolly pine plantation. Enzyme histo-chemistry of gum-resin canals of Forest Sci 17: 170-177 some members of Burseraceae. Proc Indian Natl Nair GM, Patel KR , Shah JJ (1981 a) Histological changes Sci Acad B 46:506-511 in the gum-resin producing cell system in Shah JJ, Nair GM , Kothari IL ( 1982) Ultrastructural changes Commiphora mukul Engl Induced by mechanical in the gum-resin ducts of the bark of Commiphora injury. Proc Indian Acad Sci (Plant Sci) 90: 129-136. wightii (Arnott) Bhandari induced by mechanical Nair GM, Patel KR, Subramanyam SV, Shah JJ (1981 b) injury. IAWA Bulletin n s 3:185-192 Secretion of resin across the wall of the epithelial Singh RB, Naiz MA, Ghosh S (1994) Hypolidemic and cell in the gum-resin canals of Commiphora mukul antioxidant effects of Commiphora mukul as an Engl Annals Bot 47:419-422 adjunct to dietary inpatients with Nityanand S, Kapoor NK (1971 a) Hypocholesteremic effects hypercholesterolemia. Cardiovascular Drugs of Commiphora mukul resin. Indian J Exp Bio 9:376 Therapeutic 8:659-664. Nityanand S, Kappor NK (1971 b) Cholestrol lowering activity Singh AK, Suri SS, Ramawat KG (1997) Somatic of the various fractions of Guggul . Indian J Exp Bio embryogenesis from immature zygotic embryos of 9:395-347 Commiphora wightii, woody medicinal plant. Ogbazghi W (2001) The distribution and regeneration of Gartenbauwissen 62: 44-48 Boswellia papyrifera (Del) Hochst in Eritrea. PhD Singh BB, Mishra L, Aquilina N, Kohlbeck F (2001) Thesis. Wageningen University and Research Usefulness of guggul (Commiphora mukul) for Centre Wageningen The Netherlands osteoarthritis of the knee; An experiemental case Peters CM (1996) The Ecology and Management of Non- study. Alt Therapeutic Health Med 7:112-114 Timber Forest Resources.World Bank Tech Paper Smith FG, van Jaarsveld EJ, Arnold TH, Steffens FE, Dixon No 322 World Bank Washington DC RD, Retief JA (1997) List of Southern African succulent plants UMduas Press Pretoria

6 Sterck FJ, Bongers F (2001) Crown development in tropical Ticktin T (2004) The ecological implications of harvesting rain forest trees: patterns with tree height and light non-timber forest products. J Appl Eco 41:11-21. availability. Jl Ecol 89:1-13 Torquebiau E (1984) Man-made dipterocarp forest in Sterck FJ, Schieving F, Lemmens A, Pons TL (2005) Sumatra. Agroforestry Sys 2:103-127 Performance of trees in forest canopies: Toon R, Ogbazghi W, Wessel M, Bongers F (2006) The effect explorations with a bottom-up functional-structural of tapping for frankincense on sexual reproduction plant growth model. New Phytologist 166:827-843 in Boswellia papyrifera. J Appl Eco 43:1188-1195 Sunnichan VG, Mohan Ram HY, Shivanna KR (2005) Tripathi SN, Sastri VS, Satyavati GV (1968) Experrimental Reproductive biology of Boswellia serrata, the and clinical studies of the effect of Guggulu(C source of salai guggul, an important gum-resin. Bot mukul) in hyperlididemia and thrombosis. Indian J Linnean Soc 147:73-82 Med Res 2 Tajuddin KA, Dhuhan SPS, Ram D, Thakur RS (1994) Indian Varier VPS (1994) Indian Medicinal Plants. http://www. bdellium-Commiphora wightii CROMA 16:75-86 vedamsbooks.com/no.9774.htm Thomas M, Shrivastava AK, Tomar SS (2010 a). Guggul; Wang X, Greilberge J, Ledinski G, Kager G, Paigen B, Jurgens production and conservation. Directorate of G (2004) Atherosclerosis 172:239-246 Research Services, Jawaharalal Nehru Krishi Vishwa Vidyalaya Jabalpur Thomas M, Shrivastava AK, Tomar SS (2010 b) Guggul (in Hindi) Directorate of Research Services, Jawaharalal Nehru Krishi Vishwa Vidyalaya Jabalpur

7 8 JNKVV Res J 44(1): 9-17 (2010)

Upland rice in Madhya Pradesh: Problems and Prospective

I.M. Khan, R.K. Tiwari and S.K. Rao JNKVV, College of Agriculture, Rewa (MP)

Abstract Keywords: Upland, ecology, drought, rainfed, genotype, osmotic, weeds Among the different rainfed ecologies the upland area of rice cultivation occupies 0.84 million ha. In Madhya Pradesh with Rice is the dominant staple food of India, accounting for lowest productivity varying with the variation of rainfall in more the 70% calories in take in majority of states different years. Maximum area and production in rice crop is including southern, central and eastern India. Rice at Jabalpur division followed by Rewa division. Low production needs to be raised by 50% more up to 2030 productivity in upland rice is a result of crop depending on to meet the challenge of increasing demand of rice rains mostly caused moisture stress, absence of surface water accumulation, direct seeding, heavy infestation of because of increasing in population of rice consumers. weeds, insect pests and diseases. Unavailability of short The bulk of rice area under rainfed upland or unfavorable duration improved high yielding varieties suited to rainfall for rice cultivation found in eastern India in Assam, east upland environment and poor fertility of soils also cause low Madhya Pradesh , Orissa, Bihar, Jharkand eastern U.P. productivity in upland rice. The occurrence of drought during and West Bangal. In M.P. rice is grown all most in all the crop growth period may very and occurs at any growth stage districts as a rainfed crop and occupies nearly 0.84 million however, early drought, mid season and terminal drought ha of land against the 5.23 million ha of total upland area after flowering are most common in rainfed upland rice crop. of India (Table 1). Early duration varieties having mechanisms to conserve moisture loss such is leaf rolling, leaf expansion sensitivity Table 1. Major rainfed upland area of India to low water potential at vegetative phase and panicle water potential at reproductive phase with osmotic adjustment are States Area (Million ha) important. The identification of rice germplasms and rice breeding lines (IET-numbers) have done for their suitability Bihar (+ Jharkhand) 0.51 in upland area under AICRIP and CRURRS, Hazaribag over M.P. 0.84 the years. The mean genotype yield ranged between 2.8 to Orissa 0.85 3.0 t/ha and the varieties performing better are Kalinga III, U.P. 1.65 Vandana, Heera, Annada, Turant dhan, Tulasi, Poorva, JR- W.P. 0.84 75 and N-22. The yield losses due to weeds ranged for 51 to Assam 0.54 78% with maximum density in July seeded crop. Weed control Total 5.23 efficiency can be improved with the application of early post Highly diversified climatic condition and variable emergence herbicide followed by one manual weeding at 25-30 days after sowing. The application of NPK @ 60:40:30 soil types resulted productivity not only low but also kg/ha found useful to increase grain yield with three split fluctuating in different years. The average productivity of application of nitrogen fertilizer at sowing tillering and PI stage. rainfed rice is in ranging 1.2 to 1.5 t/ha although the Brown spot, leaf blast and leaf blight diseases are most potential is munch higher (Murlidharan et al. 1988). In common in rainfed upland areas and may be control by the rainfed area it self different types of rice farming situations seed treatment with Diathan M 45 or mancozeb or Beam. existed in M.P. More than 78% rice is grown in rainfed as Among the insect pests BPH, WBPH, leaf folder and upland unbunded, bunded and bandh areas in variable Gundhibug are most common and occurred at various types of soil, topography, temperature and rainfall. stages of crop growth. Monocrotophost, Ethofenprox or acetamipride + Chloropyriphos are common insecticides used in the control of insect pests. As the rainfed upland Upland rice environment conditions are highly variable depending on soil water relationship the researchable issues are enumerated for further especial consideration for varietal development weed, In M.P. upland rice is generally broadcast seeded with water, soil and plant protection issues to harvest maximum the onset of monsoon during mid June to mid July and yield from upland ecology. harvested in September-October. Rainfall is the most

9 Table 2. Area production and productivity of rice crop in India and M.P. (1999-2004)

Years AreaLakh ha Production Lakh ha Productivity India M.P. India M.P. India M.P. 1999-2000 451.6 17.42 896.8 17.5 1990 1060 2000-2001 447.1 17.00 849.8 9.8 1900 610 2001-2002 449.0 17.80 993.4 16.9 2080 1010 2002-2003 402.8 16.80 726.5 10.3 1800 650 2003-2004 424.1 17.20 870.0 17.5 2050 1070

Table 3. Area and production of rice crop among the division of M.P.

Division of M.P. Area (000 ha) (%) Production (000 t) (%) Jabalpur 778.4 45.56 904.5 51.8 Ujjain 3.9 0.23 2.6 0.15 Sagar 160.9 9.42 107.5 6.17 Gwalior 29.5 1.73 62.8 3.60 Bhopal 62.6 3.66 64.0 3.67 Indore 52.0 3.04 41.8 2.40 Rewa 621.3 36.36 560.2 32.13 variable and least predicable component of the physical to rice crop growth besides uncertainly in rain water environment in upland rice ecosystem. 700-1200 mm availability in upland rice, the swings in the onset, annual rainfall is broadly classified as upland. The continuity and withdrawal pattern of monsoon make crop distribution, however, is more critical then total seasonal production in rainfed areas a risky proposition. rainfall (Mahapatra and Shrivastava 1984). Soils are varying in upland environment with low Rainfed upland areas received its peak fertility level, low base saturation and also in cation precipitation in July-August and dry spell of 5-15 days exchange capacity. The texture of the soils bearing sendy may occur any time during crop growth, diurnal temperature loam-loam to clay loam types. Colour changes from red- regimes also play role (AICRIP Annual Report 2008) yellow-gray in a sequence from aerobic to semi aerobic while, radiation levels during wet season is not restricted uplands. Soils are shallow is several areas and prone to

1600 1481

1400 1246

1200 1134.2 1090 987 1003.4 1000 949.8 924 885.8 852.2 831.8 800 742.5 696.8 Rainfall (mm) Rainfall

Rainfall(mm) 600

400

200

0 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 Ave rage 2008 Normal (1998- (1971- Years 2007) 2007) Yea rs

Fig.1. Rainfall (mm) in 2008 compared with mean annual rainfall (mm) of Rewa

10 Table 4. Rice zone in M.P.

Crop zones Agro-climatic Soil Rainfall range District regions type (mm) covered 1. Rice zone Chhatisgarh plains Red and Yellow 1200 to 1600 Balaghat Rice zone Northern Hill Region Red and Yellow 1200 to 1600 Shahdol, Anuppur, medium black Sidhi and parts of and medium/light Mandla 2. Wheat rice Kymore Plateau and Mixed red and 1000 to 1200 Rewa, Satna, Panna, zone Sapura Hills black soils(Medium) (Partly) Seoni and Katni soil erosion. This is particularly true for the upland common situation of upland characterizes as (I) Aerobic environment where rice is grown in a heterogonous array soils (ii) absence of surface water accumulation (iii) of physiographic, edaphic, climatic and socio- economic dependence on rain (iv) plan to slopping topography and conditions. (v) direct seeding. However, semi aerobic situations in which short duration rice, are directly seeded under dry Rice grown on both bunded and unbunded flat conditions but where water accumulates for short period lard by seeding under dry condition and that depends on of time, are also considered under upland rice (Murty et rainfall for moisture and usually refers to early maturing al. 1985). rice (80-110 days) varieties (Mutry et al. 1985). The most

Table 5. Agro climatic zones and constraints in upland rice cultivation

Zones Districts covered Major constraints Northern hills (Part) Shahdol, Umariya, i. Drought Anuppur, Singrauli, ii. Poor and deficient soil to P Mandla iii. Lack of suitable upland varieties

Kymore plateau Stpura hills Rewa, Satna, Panna, i. Low N and P2O5 soils Jabalpur, Seoni ii. Soil erosion iii. Drought iv. Haveli system exists Vindhya plateau Guna, Damoh, Vidisha, i. Lack of early duration varieties Bhopal, Raisen, Sehore ii. Drought occurs at flowering iii. Insect pests and diseases problem Centre Narmada valley Hosangabad i. Deficient soil with N and Z ii. Drought iii. Lack of early variety Grid zone Bhind Gwalior, Morena, i. Alluvial soil Shivpuri, Guna ii. Zn and Mn deficient soil iii. Drought and salinity problem iv. Temperature fluctuation Bundelkhand Datia, Tikamgarh i. Drought ii. Poor fertility soil iii. Low water retention capacity in soil iv. Lack of short duration varieties Satpura plateau Chhindwara, Betul i. Drought ii. Poor fertile soil iii. Lack of early varieties Malva plateau Mandsaur, Ujjain, Dhar, i. Drought Indore ii. Poor soil iii. Lack of early/suitable varieties Nimar valley Khandwa, Khargone i. Drought Burhanpur ii. No. use of fertilizers iii. Lack of early varieties iv. Insect pest and disease Jhabua hills Jhabua i. Lack of early varieties ii. Drought iii. Insect pass and diseases

11 Following agro climatic zones falls in M.P. at sowing and a rainfall probability such that water deficits covering various districts having several production may occur unpredictably at any growth stage but constraints of rice crop. particularly in the panicle initiation and flowering stage have different set of mechanism. With an increase probability of rainfall in the flowering stage a large crop Major constraints in upland rice in M.P. canopy at anthesis is desirable to maximum number of spikelets set per panicle. The drought recovery potential The rainfed issues are highly variable depending upon (developmental plasticity) character in upland rice is most soil water relationship. The problem of rainfed uplands desirable (Khan and Bhagat 2005). are distantly different form other ecologies and specific farming situations. The management of water/moisture In most of the districts of M.P., both unpredictable stress, plant population, weed problems and low soil and terminal drought during monsoon season are common fertility. The infestation of various insect pest and diseases (Khan and Khan 2003). Here rice varieties require are also serious concern in upland rice cultivation. mechanism that conserve moisture in the vegetative phase and that allow adaptation to water deficits as the soil The poor yield in upland rice in M.P. is also due water depleted. In general the desirable characteristics to lack of high yielding extra early- early genetic materials. of rice varieties for rainfed upland area of the region are For rainfed uplands, the varietal development preferably as follows: extra earliness starting from 70 to 90 days duration promise of 2.5 to 4.0 t/ha like Vandana and Kalinga III • Fast germination and early establishment of deep roots needed. These varieties escape drought as these are • Rapid phonological development grown during the assured rainfall period of an area. • Developmental plasticity However, tolerance to drought was incorporated to some • Leaf expansion highly sensitive to water deficit extent in the variety Vandana (Sinha et al. 1994; Prasad • Ability to adjust osmotically and Sinha 1989). • Large assimilate transfer from stem to grain • Dehydration tolerance at vegetative and grain filling stages Drought These characters are important in variety for successful rainfed upland crop and not exhaustive for There are three major mechanisms that enable plant to different patterns of drought in the region (Turner and Begg resist drought (Turner 1986 a) I. Drought escape: The 1981; Ludlow and Muchow 1990) considered resistance ability of plants to complete its life cycle before serious mechanisms in constructing an ideotype for improved yield water stress develop. The genetic materials having early under dry land condition in rice. These included drought ness characteristics may best fit in upland areas of M.P. escape and tolerance at high and low water potentials. II. Drought Torrance: This is the ability of plant to endure Different characteristics were suggested for the crop under periods of rainfall deficit while maintaining high tissue terminal, unpredictable or both drought conditions (Khan water potential. In this mechanism plant avoided tissue et al. 2005). dehydration and III. Drought tolerance: the ability of a plant to endure rainfall deficits at low tissue water potential. The occurrence of drought during crop growth period in Drought management rainfed upland may very and occurs at any growth stage. However there are four major patterns of drought i.e. early The work on characterization and identification of rice drought, terminal drought, unpredictable drought and germplasms and rice IET numbers have done in central unpredictable terminal drought (Turner and Begg 1981). eastern part of India for their suitability in upland area 151 The crop having adequate water at sowing but with a Gora rice were evaluated for 60 agro morphological declining water supply particularly after flowering (Terminal characters, 351 rice genotypes were characterized for drought). Mechanisms to conserve moisture loss such 15 quality characters and 64 upland rice varieties were as leaf rolling, leaf expansion sensitively to low water classified using an algorithm based an the alleles patent potential are required during the vegetative phase and at 5 Isozymloci. Thirty five belonged to the indica group mechanism to osomatically adjust and gradually lowering and 22 belonged to the partly traditional and partly soil and leaf water potential are needed during the improved group were identified at central rainfed upland reproductive phase (Ray Chaudhari et al. 1984; Khan and rice research station, Hazaribag (Chohan et al. 1996). Tiwari 1990). Last twenty three years records from 1985 to 2008 on In other situation crop with adequate soil moisture the performance of genotypes in the All India Coordinated

12 upland experiments results revealed that top three reducing genotypes ranged for 2.8 to 3.0 t/ha. The lowest yield was 2.0 t/ha and the highest 4.2 t/ha. Annually in rainfed upland experiments 12 to 42 elite rice cultures were tested at AICRIP, Rewa centre (1985-2008) over the years. There was a perceptible negative trend in terms of a reduction in yield levels recorded with newer genotypes developed for uplands. During these years extra early duration varieties having escaping to avoidance and early to mid early duration varieties having avoidance to resistance mechanisms with higher yield potential was released. These are Kalinga III, Vandana, Heera, Annada, Turant dhan, Tulasi, Poorva, JR-75, N 22, JR 353 etc. the catchments area or source area over which runoff is Kalinga III and Vandana showed reduction in plant height maximized and other is the storage facility or use area. and leaf area when exposed to moisture stress for 8-10 Runoff farming is basically a water harvesting system days. Both varieties exhibited higher stomatal resistance designed to provide supplemental or life saving irrigation under stress condition. The reduction in dry matter to crop during critical period of moisture stress. Water production ranging between 40 and 63%,and leaf area harvesting techniques for holding the water collected from between 50 and 55% at low water availability during stress a catchments area are of two types (a) the soil profile condition. Leaf water potential of third leaf after rolling and (b) tank or ponds. The various factors for designing of was good indicator of drought tolerance. Complete rolling water harvesting system and soil profile storage of leaf was associated with non opening of spikelets at characteristics must be considered simultaneously flowering (Chen et al. 1990; Turner and Bagg 1984). (Frasier 1987). A water harvesting pond was developed in Root thickness, an important character for drought 1991-92 at JNKVV, Kuthulia farm Rewa proved avoidance mechanism was found positively correlated with instrumental in stabilizing dry land crop production. The root dry weight and rooting depth, Rice variety Annada, water harvesting tank has capacity to accumulate 4100 N-22, Tulasi, Poornima, Vandana and Kalinga III exhibited cu.m. of water and tank received run off water from 3.20 superiority (O'Toole and Bland 1987; Khan et al. 1997) ha catchment area having the general slop @ 0.5% Root dry matter and drought score traits are tolerant towards the tank. The area under water harvesting tank character found in N-22, Annada and Kalinga III as they was 2373 sq.m. having trapezoidal shape. A total of 4719 produced more dry matter under stress condition. cu.m. water was harvested through runoff which was caused by 885 mm rainfall and at the time of recycling 3073 cu.m. water present. Out of which 2387 cu.m. water Hybride rice was recycled to 1.65 ha for Rabi land preparation. The yield of Rabi crops Gram and Coriander increased by 23 Several rice hybrids of short duration maturity range (100- and 22.14% higher in pre sowing irrigation of harvested 120 days) has bean developed after pro agro 6201 which water than no pre sowing irrigation in dry land condition are most suited for upland bunded areas of M.P. Rice (Anonymous 2002-05) hybrid JRH- 4, JRH-5, KRH-2, JRH-8, JRH-10, JRH-11 The harvested water could be utilized judiciously and PRH-10 were found most suitable for cultivation in at critical stages of the crops in the cultivated area of the M.P. with breaking the yield barrier ranging from 6-8 t/ha water shed. Irrigation from reservoir not only helpful in life (AICRIP-Rewa 2007-08) under transplanted condition. saving of crop during critical stress condition but also successfully mature the early duration rice crop (AICRP Water management on dryland 2002-2005). Water stored in ponds needs to be utilized at critical stage of growth by the most efficacious method in upland areas. In Kymore plateau sub zone region about 60% of the rainfall is lost due to run off during monsoon season. (AICRIP on dryland 2005). The runoff losses can be Fertility management reduced up to 20% by early establishment of crop. The rain water harvesting is most efficient technology in upland Water and fertilizers increases each other efficiency of areas not only to reduce run off loss of water but also to crop, low quantity of fertilizer use or no use of fertilizers conserve the water and soil. There are two basic in Rice crop is a common practice among farmers of the components of a water harvesting system. First one is

13 region during rainy season. Supply of adequate plant The, most critical period for crop weed nutrients helps in early development of crop cover due to competition in upland condition are first 30 days period. vigorous shoot and root growth. Nutrient application Echinocloa colona, Setaria gluaca, Cyprus spp. and increases water use and better biomass partitioning in Ageratum spp. are most common and exotic weeds rice (Mishra 1999; Khan et al. 2005). species have been reported from upland area of Kymore plateau and Satpura hill zone of M.P. (Choure et al. 2001; Upland soils are generally poor in fertility and Rajpoot et al. 2002). The threshold density of weeds varied can not with stand the water stress on the occurrence of with date of rice seeding. It was 27-37 plants/m2 for 3rd - drought. A well fed soil by application of manure and 4th week of June seeded rice, 52-106 for Ist week of July fertilizers could provide deep and extensive roots capable seeded rice at 85-262 plants/m2 for 2-3rd week of July of absorbing near water and nutrients from deeper layers sown crop. (Mishra 1997). Early duration rice varieties and greater volume of the soil. The application of NPK @ showed better weed competing ability due to some of the 60:40:30 kg/ha in short duration rice has been found useful trails like early emergence of plumule, initial higher LAI, to increase the grain yield. Three split doses of nitrogen tiller number plant height, biomass, number of roots/plant at sowing, tillering and panicle initiation stage found and root depth. Weeds of upland rice emerged in three beneficial because of efficient utilization of nitrogen by flushes which coincided seedling (grassy weeds), tillering the crop with minimum losses. Zinc application @ 10-15 and reproductive phase (broad leaf weeds) of crop (Choure kg Zn SO /ha in zinc deficient light textured soils of upland 4 et al. 2001). areas favours to increase the crop production.

Weed management Weeds

Normally two hand weeding at 20-25 and 40-45 days after In upland rice yield losses due to weeds ranged from 51 sowing controlled weeds satisfactorily. Continuous use to 78% and predominant weed species identified are of single herbicide led to un desirable shift in weed flora. Aeschynomene indica, Ageratum conyzoides, Commelina Pre emergence application of butachlore @ 1.5 kg a.i/ha nudifolia, Cynodon ductylon, Cyprus rotandus, Celasia followed by one hand weeding (30 DAS) produced argentea, Dactylctenium aegyptium, Digitaria ciliaris, significantly higher grain and straw yield while in early Echinocloa colona, Panicum repens, Setaria gluaca etc.

Table 6. Population and dry weight of weeds and growth characters of rice as influenced by weed control treatments

Treatment Time of Grassy Brond- Sedges/m2 Total Dry Weed application weeds/m3 leaved weeds/m2 weight of control (DAS) weeds weeds/m2 efficiency Butachlor+Safner 50EC @ 1.00* 3 20.75 21.25 18.25 60.25 93.75 75.65 (4.61) (4.66) (4.33) (7.79) Anilophos+Ethoxy sulfuron 22 SC @ 0.025+0.010* 10 21.25 11.25 22.25 54.75 78.75 79.54 (4.66) (3.43) (4.77) (7.43) Anilophos+Ethoxy sulfuron 22 SC @ 0.375+0.015* 10 16.75 10.25 16.50 43.50 61.25 84.09 (4.15) (3.28) (4.12) (6.63) Fenoxapro-p-ethyl 7.5 EC @ 0.056* 15 26.75 23.25 36.25 86.25 112.50 70.78 (5.22) (4.87) (6.06) (9.31) Fenoxapro-p-ethyl 7.5 EC @ 0.075* 15 24.25 22.75 25.75 72.75 10550 72.59 (4.97) (4.82) (5.12) (8.56) Anilophos+2,4-DEE 25+ 32EC @ 0.40+0.53* 10 19.75 13.50 26.50 59.75 90.00 76.62 (4.50) (3.74) (5.19) (7.76) Cyhalofop-butyl 10 EC @ 0.06* 10 20.25 12.75 23.75 56.75 87.50 77.27 (4.55) (3.64) (4.92) (7.57) Cyhalofop-butyl 10 EC @ 0.08* 10 18.75 10.75 16.75 46.25 68.75 82.14 (4.39) (3.35) (4.15) (6.84) Hand weeding (twlce) 20 & 40 14.25 8.25 10.75 33.25 12.00 96.88 (3.84) (2.96) (3.35) (5.81) Unweeded control - 70.75 52.25 103.75 226.75 385.00 - (8.44) (7.26) (10.21) (15.07) CD (5%) - 0.76 0.30 0.42 0.50 11.53 - Figures in parenthesis are the transformed values, *kg a.i./ha, DAS=days after sowing, Grassy weeds-Echinochloa colonmum, E. crusgalli, Eclipta alba, Eleusine indica, Setaria glauca, S. intermedia, Cynodom dactylon Broad leaved - Commelina benghalensis, Sedges - Cyperus rotundus and C.iria

14 post emergence group several herbicide were tested over spray @ 2.5 g/ha protected the crop. Seed dressing with the years and found that spray of Anilophos, Thiobencarb, Beam 75 WP (Tricyclazol) and Fongorene 50 WP pendimethalin, pretilachlore, pyrazosulfuron-ethyl (Pyroquilan) @ 3 g/kg seed reduced leaf blast significantly. oxadiazon, oxyflourben, cyhalofop and Fenoxaprop-p Besides of above technologies, integrated disease ethyh have been found to control upland rice weeds. management is also found effective. The major component (Mishra 1996; Choure et al. 2001). Weed control efficiency strategies viz., host plant resistant varieties, need based can be improved when above mentioned early post application of fungicide and cultural management are emergence herbicide were followed by one manual effective for the major upland rice diseases. weeding at 25-30 days after sowing. Evaluation of new fungicidal formulation against leaf blast of rice showed that sivic 75 WP was superior Diseases with 3566 kg/ha grain yield followed by Tricyclazol (3533 kg/ha). Among the biopesticides tricure (ie 5 ml/lit) was Infestation of rice crop by diseases and insect pests are found most promising in controlling the leaf blast and most common after weed infestation in upland increasing the grain yield (3704 kg/ha) followed by Achook ecosystem. Surveillance of diseases in upland rice was (3111 kg/ha) (AICRIP-2006). studied through AICRIP production oriented survey programme over the years. Diseases were prioritized in Insect pests the following after: (a) Upland rice (aerobic) where the cultivation of early Occurrence of rice insect pests studied through POS duration rice is most common and possible, Brown conducted by AICRIP Rewa center in the Kymore plateau spot (Helminthosporium oryzae) and leaf blast sub zone of M.P. revealed that BPH, WBPH, leaf folder (Pyricularia grisea) infestation found. and Gundhi bug are the most common insects in the (b) Upland rice (semi aerobic) where the cultivation of upland ecosystem. Case worm and GLH are also early to medium duration rice varieties are common. appearing since two - three years. Insect infestation has Leaf blast, Bacterial leaf blight (Xanthomonas been observed at all stages of crop growth. However, BHP, campestries oryzae) and Brown spot are infected the WBPH and leaf folder infested the crop during vegetative rice crop in order). and reproductive phases and Gundhi bug during flowering to grain filling stage of crop growth. Drought situation Brown spot disease was found severe during aggravated infestation of WBPH and BPH in rice crop September month while the infestation of blast disease (AICRIP-2006). are common in the month of August and September in the region (AICRP-POS 2007). Insect management Increasing level of nitrogen (0-80 kg/ha) increased disease intensity whereas, increasing level of phosphorous (0-50 kg P O /ha) and potassium (0-30 kg Spraying of dimethoate, oxydemeton-methyl or 2 5 ethofenprox @ 0.04% or dusting of malathion/quinalphos K2O/ha) reduced it. Plant height, number of filled grains and panicle weight were significantly and negatively @ 1.5 kg ai/ha control the insect pests. Acetamipride correlated with disease intensity (Shukla et al. 1996). 0.4% + chlorphyriphos 20 EC found significantly superior Local genotypes and improved high yielding varieties to control Gundhi bug. The maximum population of case exhibited susceptibility to these diseases but losses were worm and gundhi bug were found (129 and 99 less due to low level of fertilization in the farmers field. insects/week respectively) in 3rd week of October when Moisture stress effect disease development indirectly by temperature was 29.70c and RH 88%, bright sunshine predisposing the host as intermittent drought spells are hour (8.0) and no rainfall (AICRIP-2007). frequent in upland rice, pre infection moisture stress is important epidemiologically. Future strategies

Disease management The research issues of rainfed upland are highly variable depending on soil-water relationships, plant type for rainfed The upland rice diseases can be managed using host uplands, management of plant population, weeds, water plant resistant varieties. Seed treatment with Diathan M-45 and soil need special consideration. For the rainfed upland or Indofil M- 45 Mancozeb @ 0.3% followed by need based the varietal development programme profitably utilized the concept of extra early ness. These varieties starting from

15 Issues Strategies Low productivity in rainfed upland ecologies Developing high yielding varieties suited to different rainfed ecologies employing both conventional and molecular approaches Designing New Plant Type varieties with good grain quality involving tropical Japonica and indica parents for shallow low lands Developing high yielding hybrids for rainfed situations with incorporated insect and disease resistance and good grain quality Continued yield losses due to abiotic stresses Breeding for abiotic stress tolerance using conventional and like drought molecular tools Resource utilizationmobilization and Developing ICM constituting improved stand establishment conservation under : i. Hybrid rice suitable cropping geometry and efficient fertilizer, water and ii Kharif rice weed management. Water saving irrigation Breeding and evaluation of variety, aerobic rice, SRI and alternate wetting and drying technique. Risk alleviation for abiotic stresses Soil and crop management under drought. Enhancing cropping intensity Rice based multiple cropping system including intercropping sequence cropping, relay cropping under different rice ecologies. Nutrient use efficiency Innovative formulation/modification of fertilizers for macro and micronutrients, site specific and plant nutrient demand based nutrient application innovative methods of placement/application. Soil quality improvement and resilience Sustainable nutrient management by optimizing nutrient addition and minimizing nutrient losses, improving physical and biological health of soil through INM, study of substrate dynamics and carbon sequestration Organic rice Crop nutrition, crop protection and grain quality improvement through organic/natural resources management, bio-control of pest including use of bioagents. Productivity enhancement of rice based Zero tillage/reduced tillage and efficient water management to cropping systems by improving soil physical improve soil structure, water storage and transmission properties properties to enhance crop productivity in rice pulse/oil seed cropping system. Declining water availability for rice cultivation • Increasing water productivity in rice based cropping system in rainfed as well as irrigated ecology through innovative • Water management technology for aerobic rice. Host plant resistance Identification of resistant/tolerant donors having high yield potential, against major insect pests and their utilization for varietal improvement Host plant resistance to major rice diseases Screening of rice germplasm, wild rices and collaterals hosts for identification of source of resistance to blast, brown spot, sheath blight , sheath rot, false smut, bacterial blight and RTD. Physiology and biochemistry of abiotic stress • Collection and screening of rice germ plasm with tolerance response in rice plants to various abiotic stresses • Working of the mechanisms of abiotic stress tolerance. Poor adoption of rice technology Constraint analysis and assessment of technologies will provide insight into the issue. Accordingly, demonstrations, OFT, Trainings and other extension methods will be focused to suit the farmers.

70 to 90 days showed a promise of 2.5 to 4.0 t/ha. These varieties escape drought as these are grown during the competitiveness in upland rice varieties , management of assured rainfall period of the area. The work on drought weeds through replacement of broadcasting method with tolerance coupled with higher yield, resistance/ tolerance line sowing are the strategies to be perused positively. to blast, brown spot, and gundhi bug will have to be The research issues relating to crop production, intensified based on the leads obtained so far. Concept economic viability and environmental quality improvement of crop varieties mixtures, inter and sequence cropping are enormous in rainfed systems. To deal with such systems require more attention. Incorporation of weed issues, capital-intensive research, efforts are necessary,

16 the implementation of which will help to generate capital Khan IM, Khan AA, Tiwari KL (1997) Root characters and saving technologies for resource poor farmers. Besides, their correlations with grain yield in traditional rice the input intensive technology options will also be made genotypes under rainfed upland conditions. JNKVV available for progressive farmers. Some important issues Res J 31(1&2):15-19 and their strategies are as follows. Khan IM, Tiwari KL (1990) Performance of rainfed upland rice in relation to drought. Indian J Dryland Agric Res & Dev (1&2):89- 92 References Ludlow MM, Muchow RC (1990) A critical evaluation of trails for improving crop yield in water limited environment. Adv in Agro 43:107-149 Anonymous (2008) Annual progress report -2007-08: All India Coordinated rice improvement project (ICAR), Mahapatra IC, Shrivastava VC (1984) Management of upland JNKVV College of Agriculture Rewa. MP rice. Problems and prospects. Oryza 21(1&2):20-32 Anonymous (2005) Annual progress report-2002-05 AICRP. Mishra GN (1999) Fertilizer use in upland rice. Fertilizer News Dryland Agriculture (ICAR) JNKVV College of 44(7):29-36 Agriculture Rewa MP Mishra GN (1997) Competitive ability of upland rice cultivars Anonymous (2001) Annual progress report-1990- with weed and its utilization in weed control. Oryza 2001.Directorate of Rice Research Rajendranagar 34(2):125-130 Hyderabad India Mishra GN (1996) Herbicidal control of upland rice weeds its Anonymous (2008) Annual progress report-2007-08. limit and impact on agro-eco system. Farmers and Directorate of Rice Research, Rajendranagar Parliament 31(3):16-30 Hyderabad Muralidharan K, Shide JE, Pasalu IC, Venkataraman SV, Prasad ASR, Kalode MB, Rao AV, Reddy APK (1988) Chen CC, Lui LM, Hua M (1990) Differences in leaf water Rice in eastern India- a reality. Oryza 25:213-245 potential in rice genotypes with different drought tolerance. Acta Agric Univ Perkinensis 16:45-58 Murty KS, Chandra D, Srinivasulu K (1985) Upland rice Choure JP, Namdeo KN, Tiwari RK (2001) Efficacy of environment in India and fitness of improved herbicides in direct seeded rice under paddled technologies in progress in upland rice research conditions. Ann Pl Soil Res 3(2):323-325 IRRI Philippines pp 25-34 Chauhan JS, Moya TB, Singh RK, Singh CV (1996) Growth O'Toole JC Bland WL (1987) Genotypic variation in crop and development under different soil moisture plants root systems. Adv in Agro 41:91-145 regimes in upland rice (Oryza sativa). Ind J Plant Prasad K, Sinha PK (1989) Upland rice varieties for plateau Physiol 39(4):270-272 region of Bihar. Indian Farming 39(2):5-6 Frasier GW (1987) Water harvesting for collection and Rajpoot PK, Namdeo KN, Tiwari RK (2002).Development of conserving water supplies: in proceeding of the suitable production technology for direct seeded rice consultants workshop on the state of the art and under puddled condition. Ann Pl Soil Res management: Alternatives for optimizing the 4(1):138-140 productivity of SAT Alfisoils and related soils. pp Raychaudhuri NCB, O' Toole JC, Cruz RT, Padilla J (1984) A 67-77 1-3 Dec 1983 ICRISAT Patancheru AP India rapid method for determining plant water stress in Khan IM, Bhagat DV (2005) Influence of weather on growth upland rice. IRRI Newsletter 9(3):9 and yield parameters of upland rice. Ann Plant Shukla VD, Variar M, Chouhan JS, Maiti D, Singh RK (1996) Physiol 19(1):27-30 Competitive evaluation of seedling growth Khan IM, Bhadauriya UPS, Khan RA (2005) Partitioning of characters of brown spot infected seed in selected dry matter in early rice during grain filling phase genotypes of upland rice. Seed Res 24(1):71-72 under rainfed upland ecosystem. J Soc Sci & Hum Sinha PK, Variar M, Singh CV, Prasad K, Singh RK (1994) A Sci 4(1):73-77 New upland rice variety "Vandana" for Bihar plateau. Khan IM, Khan RA (2003) Correlation of flowering with yield Indian farming 44(3):1-3 parameters of traditional rice (Oryza sativa L.) Turner NC (1986) Adaptation to water deficits: A changing genotypes of Kymore plateau zone under rainfed perspective. Australian J Pl Physio 13:175-190 upland condition. Indian J Applied and Pure Biol Turner NC, Begg JE (1981) Plant water relation and adaptation 18:53-56 to stress. Plant and Soils 58:97-13

17 18 JNKVV Res J 44(1): 19-24 (2010)

Production, purification and characterization of phytase from Bacillus subtilis

Namrata Verma, Keerti Tantwai, L.P.S. Rajput, Sunil Kumar, Iti Gontia and Sharad Tiwari Biotechnology Centre Jawaharlal Nehru Krishi Vishwa Vidyalaya Jabalpur 482 004 (MP)

Abstract Phytase activity has been detected in a variety of microbial systems. It is frequently found in fungi particularly in Aspergillus species like A. ficuum, A. niger B. subtilis NCDC-070 and B. subtilis NCIM-2712 were used for the production of phytase using four substrates i.e. wheat and A. terreus. Among yeasts, extra cellular phytase has bran, rice bran, soy hull and chickpea hulls. Among the four been reported in Schwanniomyces castellii, S. cerevisiae substrates, wheat bran was found to produce the optimum and Arxulla adeninivoran. In case of bacteria, phytase yield of phytase (118.23 U/mg protein form B. subtilis NCDC activity has been found to be abundant in Bacillus species 070 and 133.52 U/mg protein form B. subtilis NCIM 2712). like Bacillus subtilis, Bacillus amyloliquefaciens, Purification of the phytase produced using the selected Lactobacillus sp., Entrobacter sp., Escherichia coli, substrate samples was done after the process of dialysis. Klebsiella sp. etc. Phytases are of interest for The activity of purified enzyme was assessed under different biotechnological applications, especially for the reduction experimental conditions viz., pH and incubation temperature of phytate in food and feed stuffs. Supplementation of and period. Purified enzyme showed optimum phytase activity at a pH of 5, incubation temperature of 300C and incubation animal feedstuffs with phytase increases the bioavailability period of 20 min. The molecular mass of the purified phytase of phosphate and also reduces phosphate pollution. was approximately 40 kDa by SDS polyacrylamide gel Phytases also diminish anti-nutritional effects of food electrophoresis. having higher content of phytate. Because of its great industrial importance, two bacterial strains of B. subtilis Keywords: Phytase, Bacillus subtilis, characterization, producing novel phytases were selected for further production, purification investigation keeping in view the earlier findings reported in our laboratory. In order to achieve the higher recovery Myo-inositol hexakisphosphate phosphohydrolase, and better quality of phytase enzyme, various carbon phytase, is referred to as acid phosphatase which is mainly sources were used as substrates for the growth of capable of hydrolyzing phytate to a series of lower microorganisms by optimizing different variables during phosphate esters myoinositol and phosphate (Greiner et the process of fermentation. al. 1997). Phytases are widely distributed in plants, microorganisms and certain animal tissues. Monogastric animals such as pig, poultry and fish are not able to Material and methods metabolize phytic acid since they have only low levels of phytase activity in their digestive tracts (Wyss et al. 1998), The seeds of commercial variety of soybean (JS 335) so inorganic phosphate is added to their diets in areas of and chickpea (JG 130) for were collected from the intensive livestock production (Kerovuo et al. 1998). Phytic Department of Plant Breeding and Genetics, College of acid is having both beneficial and deleterious effects on Agriculture, JNKVV, Jabalpur. Other raw materials viz., human and animal nutrition. The literature survey indicates wheat bran and rice bran were collected from the Livestock that it has anticancer properties and preventive effects Farm, JNKVV, Jabalpur. Wheat bran, rice bran, chickpea against heart disease and diabetes. Phytic acid generally hull and soy hull were later obtained by ground to fine forms an insoluble complex with nutritionally important powder using an electrical grinder to pass through metals such as calcium, zinc, magnesium and iron, 40-mesh size sieve. The powdered substrates were then thereby decreasing their bioavailability (Greiner et al. packed in air tight glass bottles and stored in the room 1997). Its chelating property however would be alleviated conditions until further used as per the requirement of the by phytase. experiments. The enzyme producing micro-organisms, Bacillus subtilis NCDC-070 and Bacillus subtilis

19 NCIM-2712 were obtained from NDRI, Karnal, Haryana acetone:5N H2SO4:10mM ammonium molybdate in the and National Chemical Laboratory, Pune, respectively. ratio of 2:1:1, v/v/v and thereafter 100 ml of 1M citric acid was added to the assay mixture. Optical density was measured at 355 nm for all the samples prepared at five Growth of the bacterial strains different incubation periods using four substrates and the two bacterial strains of Bacillus subtilis NCDC-070 and The bacterial strains were cultured in the Luria Bertani Bacillus subtilis NCIM-2712. The activity of the enzyme medium having adjusted pH to 7.0. The cells were grown was expressed as one mmol phosphate liberated per at a temperature of about 370C for 48 h. minute.

Production of bacterial phytase Purification of crude extract

Various substrates i.e. wheat bran, rice bran, soy hulls Purification was done at 160C by the gel filtration and and chickpea hulls were taken separately and processed column chromatography after the dialysis of the samples by autoclaving 25 g of the substrate in 250 ml of distilled as per the method adopted by (Sutardi and Buckle 1988). water at 1200C for 60 min, and thereafter subjected to The culture filtrate was collected from two different strains cooling and squeezing it through the muslin cloth. cultivated on wheat bran. Five ml of the culture filtrate Substrate medium was prepared by adding was purified by gel filtration using Sephadex G-50 (1.5

0.04g-(NH4)2SO4; 0.02g-MgSO4.7H2O; 1g-Casein; 0.05g- cm x 50 cm) to a height of about 35 cm and DEAE-

KH2PO4 and 0.04g-K2HPO4 in the 100 ml of substrate Cellulose chromatography (1.5 cm x 50 cm) to a height extract and the pH of medium was adjusted to 6. It was of about 44 cm. The crude extracts of phytase of the two autoclaved and allowed to cool. Under sterilized condition, strains of B. subtilis were purified 5 fold each. 10 ml of medium of four different types was dispensed, individually, in five flasks and 0.2% of CaCl solution was 2 Assessment of phytase activity in the purified sample added to each flask. Inoculated flasks were incubated at obtained using wheat bran with B. subtilis NCDC-070 and 5 different incubation periods i.e. 24, 48, 72, 90 and 96 h NCIM-2712 on a rotary shaker (220 rpm) at 280C. Each flask containing cell culture was homogenized through Branson Sonifier. The samples were then centrifuged at 10000 rpm for 10 Phytase activity was determined using the procedure min at 40C and the supernatant was collected and stored adopted by (Greiner et al. 1997) followed by ammonium at 40C. molybdate method described by (Heinonen and Lahti 1981). The enzymatic reactions were started by the addition of 50 µl of the enzyme solution and 350 µl of Protein Estimation 0.1M acetate buffer (pH 5) containing 500 nmol of sodium phytate. The reaction mixture was then incubated at Total protein concentration of the samples were different incubation temperatures i.e.15, 20, 25, 30 and determined by Coomassie blue R-250 dye binding using 35°C for 30 min. To assess the effect of pH on phytase bovine serum albumin as a standard, as prescribed by activity, the enzyme was treated with the substrate (Bradford 1976). dissolve in acetate buffer of different pH (4.5, 5, 5.5, 6 and 6.5) and incubated at 30°C. Further at optimum pH of 5 and incubation temperature of 30°C, the samples Enzymatic activity were incubated for different time periods i.e.15, 20, 25, 30 and 35 min and the liberated inorganic phosphate was Phytase activity was determined, in all the samples measured by a slight modification in the ammonium produced by two strains using the procedure adopted by molybdate method. In the assay mixture, 1.5 ml freshly

(Greiner et al. 1997). The assay mixture for phytase prepared solution (acetone: 5N H2SO4: 10mM ammonium determination consisted of 50 µl of the enzyme solution, molybdate in the ratio of 2:1:1) and 100 µl of 1M citric 350 µl of 0.1M acetate buffer, pH 5 containing 500 nmol acid were added. The absorbance of the color developed of sodium phytate. It was incubated at 300C for an was read at of 355 nm using Thermospectronic incubation period of 30 min. The liberated inorganic Spectrophotometer Spectronic 20D+. One unit of enzyme phosphate was measured by a slight modification in the activity is defined as the amount of enzyme required to ammonium molybdate method (Heinonen and Lahti 1981) release 1 mg of phosphorus per hour at given temperature which contained a freshly prepared solution (1.5 ml) of and pH.

20 Table 1. Production of phytase (Unit/mg of protein) from different substrates at different incubation periods using B. subtilis

Incubation period Substrate B. subtilis 24h 48h 72h 90h 96h Wheat Bran NCDC-070 36.95 39.23 118.23 84.45 118.23 NCIM-2712 34.16 41.84 133.52 96.21 88.01 Rice Bran NCDC-070 40.32 52.81 96.21 96.21 96.21 NCIM-2712 54.35 51.37 95.06 95.06 95.06 Soy Hulls NCDC-070 47.62 34.13 58.73 45.65 37.33 NCIM-2712 22.52 38.09 72.53 38.90 46.79 Chick Pea Hulls NCDC-070 56.78 62.03 69.76 44.53 56.67 NCIM-2712 47.51 53.73 64.86 56.87 34.22

Characterization production of phytase and hence the enzyme so produced was taken up for the further studies on the purification using the methods of gel filtration with Sephadex G-50 The molecular weight of the purified phytase was and column chromatography with DEAE Cellulose. determined using SDS-Polyacrylamide Gel Electrophoresis as per the method adopted by (Laemmli 1970). Purification of the phytase from two strains of Bacillus subtilis Results and Discussion The results of the purification of crude enzyme extract are presented in Table 2. The purified enzyme obtained Production of phytase using two strains of Bacillus subtilis from B. subtilis NCDC-070 and B. subtilis NCIM-2712 showed similar values of specific activity of 6.8 U/mg of Four substrates i.e. wheat bran, rice bran, soy hulls and protein. Powar and Jagannathan (1982) and Kerovuo et chickpea hulls were used for phytase production by B. al. (1998) reported relatively higher values of specific subtilis NCDC-070 and B. subtilis NCIM-2712. The data activity of 8.53 and 29 U/mg of protein using the strains obtained on the production of phytase at different of B. subtilis, respectively. Likewise, the phytase of incubation periods have been presented in Table 1. The Bacillus sp. DS11 had a specific activity of 20 U/mg of results revealed that the two strains of Bacillus subtilis protein reported by Kim et al. (1998). Literature survey NCDC-070 and Bacillus subtilis NCIM-2712 when grown also revealed some variation in the values of specific on the four different substrates at five different incubation activity, recovery percentage and purification folds in the periods (24, 48, 72, 90 and 96 h) showed variations in the purification process of phytase. In the present phytase yield and these two strains recorded the investigation, the low specific activity might be due to maximum activity at an incubation period of 72 h. The less purification (5 folds). wheat bran showed maximum phytase production at 72 h of incubation period (Powar and Jagannathan 1982). Effect of pH, temperature and incubation period on the Several reports have also been published in literature on activity of the purified enzyme the yield of phytase using different strains of bacteria and showed maximum activity at a temperature of 370C and incubation period of 48 h (Popanich et al. 2002; Dharmsthiti The data on the effect of pH, temperature and incubation et al. 2004). Phytase yield was maximum within 7 days period on the phytase activity produced from two strains of incubation period by using wheat bran, mustard cake, of B. subtilis are presented in Fig 1, 2 and 3. cowpea meal, groundnut cake, coconut cake, cotton cake and black bean flour (Mandviwala and Khire 2000). Effect of pH Likewise, the commercially available oil cakes such as coconut oil cake, sesame oil cake, palm kernel cake, groundnut oil cake and cotton seed oil cake gave maximum The observations recorded in this experiment showed that phytase yield at an incubation period of 72 h (Ramchandra the optimum phytase activity was observed at pH of 5 et al. 2005). Based on the findings of above experiment, (Fig. 1) in the purified extract obtained from both the strains wheat bran was found to be the best substrate for the of Bacillus subtilis with 69.10 and 62.5 U/mg of protein

21 Table 2. Purification of phytase produced from B. subtilis

Purification stage B. Total Total Total Specific Recovery Purification subtilis volume protein activity activity (%) fold (ml) (mg) (units) (units/mg protein) Culture Filtrate NCDC-070 10 1.88 6.8 3.6 100 - NCIM-2712 10 2.38 7.4 3.1 100 - Gel filtration in Sephadex G-50 NCDC-070 5 1.15 5.1 4.4 74.9 4 NCIM-2712 5 1.57 6.04 3.8 81.5 4 DEAE Cellulose column chromatography NCDC-070 10 0.477 3.2 6.8 46.7 5 NCIM-2712 10 0.568 3.9 6.8 52.6 5 for the purified enzyme from B. subtilis NCDC-070 and (Quan et al. 2002; Cho et al. 2003; Quan et al. 2004). NCIM-2712, respectively. Several workers have also The variation observed in the values of incubation reported the pH optima at around 5.0 for bacterial temperature optima in the present investigation might be phytases (Powar and Jagannathan, 1982; Yanke et al. due to the variation in the substrate taken, fermentation 1999; Vohra and Satyanarayan 2001; Angelis et al. 2003; condition and the type of strains used in the bioconversion. Dharmsthiti et al. 2004). Contrary to this, reports have also been published for the pH optima for maximum Effect of incubation period phytase activity in the range of 5.5 to 8.5 (Kim et al. 1998; Kerovuo et al. 1998; Choi et al. 2001; Yoon et al. 1996). The variation pH optima might be due to the variation Data presented in Fig. 3 indicate that the phytase of the in the fermentation technique and also the purification two strains of Bacillus subtilis (NCDC-070 and NCIM- procedure followed for removal of impurities present in 2712) showed their optimum activity with an incubation the crude extract of phytase. period of 20 min with the values of 86.7 U/mg of protein and 69.1 U/mg of protein, respectively. The maximum phytase activity with 10 min of incubation has been Effect of temperature reported by (Kim et al. 1998). The variation in results obtained in the present investigation might be due to The optimum values of phytase activity for the two strains fermentation variables, genetic make up of the strains of Bacillus subtilis NCDC-070 and NCIM-2712 were used etc. obtained as 68.13 and 68.66 U/mg of protein, respectively at 300C (Fig. 2). Several reports have also been published Characterization of extracellular phytase enzyme from in the literature on the effect of incubation temperature on Bacillus subtilis the activity of purified phytase (Yoon et al. 1996; Greiner et al. 1997; Vohra and Satyanarayan 2002; Wang et al. 2004). These workers have reported the optimum The molecular mass and homogeneity of the purified incubation temperature in the range of 50-600C for enzyme assessed by 10% SDS-Polyacrylamide gel maximum activity of phytase. The findings of present electrophoresis (PAGE) under denaturing conditions investigation showed a relatively lower range of incubation revealed a single phytase/ protein band after staining the temperature (300C) for maximum phytase activity in gel with Coomassive. This indicates that the phytase could comparison to the reported values in the range of 40-600C be regarded as homogeneous. The molecular mass of

22 070 2712

070 118.23

2712 133.52

two purified phytases from two different strains of B. subtilis (NCDC-070 and NCIM-2712) was approximately 40 kDa (Fig. 4). Likewise, 44 kDa molecular mass of 5.0 Bacillus phytase in SDS-PAGE reported by (Kim et al. 300 20 1998 and Choi et al. 2001). 41.7 kDa molecular mass phytase produced by the soil bacterium Klebsiella 40 pneumoniae (Wang et al. 2004). The variation in molecular mass in the present investigation might be due to genetic make up of the strains used. References

Angelis MD, Gallo G, Corbo MR, McSweney PLH, Faccia M, Giovine M, Gobbetti M (2003) Phytase activity in sourdough lactic acid bacteria: purification and characterization of a phytase from Lactobacillus sanfranciscensis CBI. Int J Food Microbiol 87:259-70 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizaing the principle of protein-dye binding. Ana Biochem 72:48-254 Cho JS, Lee CW, Kang SH, Kee JC, Bok JD, Moon YS, Lee HG, Kim SC, Choi YJ (2003) Purification and characterization of a phytase from Pseudomonas Fig. 4. SDS PAGE of purified phytase from Bacillus syringae MOK1. Curr Microbiol 47:0290-4 subtilis Choi YM, Suh HJ, Kim JM (2001) Purification and properties of extracellular phytase of Bacillus sp. KHU-10. J Conclusion Protein Chem 20(4):287-92 Among the four substrates, wheat bran showed the Dharmsthiti S, Chalermpornpaisarn S, Kiatiyanjarn M, highest production of phytase from the two strains of Chanpokapaiboon A, Klongsithidej Y, Techawiparut Bacillus subtilis. Both the strains of B. subtilis (NCDC- J (2004) Phytase production from Pseudomonas 070 and NCIM-2712) were found to be the equally effective putida harbouring Escherichia coli appA. Process in the production of the phytase. Purification of phytase Biochem 40:789-93 using the techniques of gel filtration and column Greiner R, Haller E, Konietzny U, Jany KD (1997) Purification chromatography resulted in specific activity of 6.8 U/mg and characterization of a phytase from Klebsiella terrigena. Arch Biochem Biophy 341:201-206 of protein from the five folds as purification factor. The Heinonen JK, Lahti RJ (1981) A new and convenient molecular mass of the purified phytase obtained from the colorimetric determination of inorganic two strains was approximately 40 kDa. orthophosphate and its application to the assay of inorganic pyrophosphatase. Ana Biochem Acknowledgement: The authors wish to thank Director 113:313-7 Research Services, JNKVV, Jabalpur for providing all the Kerovuo J, Lauraeus M, Nurminen P, Kalkkinen N, Apajalahti facilities. J (1998) Isolation, characterization, molecular gene

23 cloning and sequencing of a novel phytase form Vohra A, Satyanarayana T (2001) Phytase production by the Bacillus subtilis. Appl Environ Microbiol 64:2079-85 yeast Pichia anomala. Biotechnol Lett 23:551-4 Kim YO, Kim HK, Bae KS, Yu JH, Oh TK (1998) Purification Vohra A, Satyanarayana T (2002) Purification and and properties of a thermostable phytase form characterization of a thermostable and acid stable Bacillus sp. DS11. Enz Micro Tech 22:2-7 phytase from Pichia anomala. W J Microbiol Laemmli U (1970) Cleavage of structural proteins during the Biotechnol 18:687-91 assembly of the head of bacteriophage T4. Nature Wang X, Upatham S, Panbangred W, Isarangkul D, 227:680-5 Summpunn P, Wiyakrutta S, Meevootisom V (2004) Mandviwala TN, Khire JM (2000) Production of high activity Purification, characterization, gene cloning and thermostable phytase from thermotolerant sequence analysis of a phytase form Klebsiella Aspergillus niger in solid state fermentation. J Indus pneumoniae subsp. Pneumoniae XY-5. Science Microbiol Biotechnol 24:237-43 Asia 30:383-90 Popanich S, Klomsiri C, Dharmsthiti S (2002) Thermo-acido- Wyss M, Brugger R, Kronenberger A, Remy R, Fimbel R, tolerant phytase production from a soil bacterium Oesterhelt G, Lehmann M, Vanloon APGM (1998) in a medium containing rice bran and soybean meal Biochemical characterization of fungal phytases extract. Biores Tech 87:295-8 (Myo-Inositol Hexakisphosphate Phospho- Powar VK Jagannathan V (1982) Purification and properties hydrolases): Catalytic properties. Appl Environ of phytate specific phosphatase from Bacillus Microbiol 367-73 subtilis. J Bacterio 151:1102-8 Yanke LJ, Selinger LB, Cheng KJ (1999) Phytase activity of Quan CS, Fan SD, Zhang LH, Wlang YJ, Ohta Y (2002) Selenolmenas ruminantium: a preliminary Purification and properties of a phytase form characterization. Appl Microbiol 29:20-5 Candida krusei WZ-001. J Biosci Bioengi 94:419-25 Yoon SJY, Choi YJ, Min HGK, Cho KK, Kim JW, Lee SC, Jung Quan CS, Tian WJ, Fan SD, Kikuchi J (2004) Purification and YH (1996) Isolation and identification of phytase- properties of a low-molecular-weight phytase from producing bacterium, Enterobacter sp. H, and Cladosporium sp. FP-1. J Biosci Bioengi 97:260-6 enzymatic properties of phytase enzyme. Enz Micro Tech 18:449-54 Ramachandran S, Roopesh K, Nampoothiri KM, Gzakacs G, Pandey A (2005) Mixed substrate fermentation for the production of phytase by Rhizopus sp. using oil cakes as substrates. Process Biochem 40:1749-54 Sutardi M, Buckle KA (1988) Characterization of extra and intra-cellular phytase from Rhizopus oligosporus used in tempeh production. Int J Food Microbiol 6:67-79

24 JNKVV Res J 44(1): 25-29 (2010)

Production of phytase from soil isolates of Pseudomonas sp. using agricultural byproduct substrates

Yuvraj Soni, Keerti Tantwai, L.P.S. Rajput, Sunil Kumar, Iti Gontia and Sharad Tiwari Biotechnology Centre Jawaharlal Nehru Krishi Vishwa Vidyalaya Jabalpur 482 004 (MP)

Abstract frequently found in fungi particularly in Aspergillus species like A. ficuum, A. niger and A. terreus. Among yeasts, extracellular phytase has been reported mainly in S. The phytase producing micro-organism was isolated from soil and identified as Pseudamonas sp. on the basis of cerevisiae. The ruminants digest phytic acid through the various microbiological test viz., Gram staining, endospore action of phytases produced by the anaerobic gut fungi test, catalase test and motility test. Identified isolate was and bacteria present in their rumenal microflora. However, used for the production of phytase using different agricultural monogastric animals such as pig, poultry and fish utilize byproducts like wheat bran (WB) as a carbon source and phytate phosphorus poorly because they are deficient in neem oil cake (NOC), cotton seed oil cake (CSOC), gastrointestinal tract phytases. Therefore, supplemental groundnut oil cake (GOC) and jatropha oil cake (JOC) as inorganic phosphate is added to their feed to meet the nitrogen sources in the substrate. Ten various substrate phosphate requirement and to ensure good growth. The combinations viz., WB+NOC, WB + CSOC, WB + GOC, WB + problem associated with phytate phosphorus utilization JOC in 1:1 ratio alongwith WB + NOC+ CSOC, WB + NOC + GOC, WB + NOC + JOC, WB + CSOC + GOC, WB +CSOC + could be solved by hydrolysis of phytate using JOC and WB + GOC + JOC in 2:1:1 ratio were made and supplemental phytase in the feeds of monogastric used for phytase production using isolate Pseudomonas animals. Therefore, phytase has become an important sp. at 370C, pH of 7 and incubation period of 48 h. Wheat industrial enzyme and is the object of extensive research. bran (WB) and neem oil cake (NOC) in 1:1 ratio of substrate There has been an increased exploitation of organic combination was found to be the best resulting in the highest residues from various sectors of agriculture and industries phytase yield from Pseudomonas sp. Various incubation over the past few decades. Crop residues such as bran, periods viz., 44, 46, 48, 50 and 52 h were maintained to know husk, bagasses, fruit seeds and oil cakes are utilized as the optimum fermentation condition for maximum phytase potential raw materials in bioprocesses as they provide yield from Pseudomonas sp. The results revealed that Pseudomonas sp. gave the maximum phytase activity of an excellent substratum for the growth of microorganism 1.102 U/ml at an incubation period of 48 h. The purification of supplying the essential nutrients to them (Ramachandran crude phytase was done in three steps beginning with et al. 2007). There is an abundance of such low cost acetone precipitation followed by gel filtration with Sephadex materials in the state of Madhya Pradesh, which could G-50 and DEAE-Cellulose chromatography. The crude extract be utilized for production of microbial industrial enzymes of phytase was purified upto 4.64 fold, showing specific such as phytase. Because of its great industrial activity of 2.71 U/ml with 34.48% of enzyme recovery. The importance, the present investigation was undertaken to purified phytase was characterized by SDS-PAGE and isolate and identify efficient microbial strains present in showed 29 kDa of molecular weight. soil, which might be of use to produce phytase with Keywords: Phytase, phytate, production, wheat bran, different combinations of substrates. substrate, Pseudomonas sp. Material and methods Phytase is widely present in nature, occurring in microorganisms, plants as well as in some animal tissues. Phytase (myo-inositol-hexakisphosphate phospho- The soil samples were collected from the fields of hydrolase) hydrolyses phytic acid to myo-inositol and ashvagandha-nilgiri and aloe vera grown in medicinal phosphoric acid. In case of bacteria, phytase activity has garden of JNKVV, Jabalpur. After this isolation, screening been found to be abundant in B. subtilis, B. and identification of microorganisms were carried out for amyloliquefaciens, Pseudomonas sp. However, it is also use in the biosynthesis of phytase. Neem oil cake (NOC),

25 cottonseed oil cake (CSOC), groundnut oil cake (GOC) under sterilized condition. 10ml of medium of ten different were procured from the local market of district Khargone, substrate combinations was taken in triplicates in 50 ml M.P. Wheat bran (WB) was collected from local market tubes and final concentration of 2% was maintained by of Jabalpur whereas Jatropha oil cake (JOC) was obtained adding CaCl2 solution in all the tubes. Now, single colony from the Department of Plant Breeding and Genetics, was isolated from these culture plates of identified two College of Agriculture, JNKVV, Jabalpur. These substrates bacterial strains. All the ten tubes were incubated for 48 were used as carbon and nitrogen sources for the growth h in incubator shaker maintaining rotation of 230 rpm and of micro-organisms. temperature 370C.

Screening and isolation of phytase producing Selection of best substrate combination and optimization microorganisms of the incubation period for phytase production

One g of soil sample was collected from each After incubation period of 48 h the supernatant was ashvagandha-nilgiri and aloe-vera field. These samples collected from the each substrate combination and were suspended separately in 10 ml of sterilized water analyzed for the content of protein and phytase activity and spread onto phytase screening plates containing 1.5% (U/ml). The best substrate combination was further used

D-glucose, 0.5% calcium phytate, 0.5% NH4NO3, 0.05% for the maximum yield of phytase along with the

MgSO4.7H2O, 0.05% KCl, 0.001% FeSO4.7H2O, 0.01% optimization of incubation period, ranging from 44, 46, 0 MnSO4 and agar 1.5%, pH 7.0 and incubated at 37 C for 48, 50 and 52 h using bacterial isolate. 16 h. After incubation, single bacterial colony was selected for pure culture bacterial strain in the same Extraction of crude enzyme medium. Identification of microorganisms was done upto genus level using different microbiological techniques viz., Gram staining, catalase test, motility test, endospore test, Different cell cultures were homogenized using Branson etc and results were compared with Bergey's manual Sonifier after attaining the growth of bacterial strain. The (Gibson and Garden 1974). samples were aseptically transferred to 15ml tubes and centrifuged at 10,000 rpm for 10 min at 40C and then the supernatant of each sample was collected and stored at Biosynthesis of phytase 40C.

The production of phytase was carried out by using Enzymatic activity different identified bacterial strains with different combinations of carbon (WB) and nitrogen sources (NOC, CSOC, GOC, JOC) in the varied ratios (WB:NOC, Enzyme activities of samples were determined by the WB:CSOC, WB:GOC and WB:JOC in 1:1 ratio whereas method described by Greiner et al. (1997) and estimation WB:NOC:CSOC, WB:NOC:GOC, WB:NOC:JOC, of inorganic phosphate was carried out by ammonium WB:CSOC:GOC, WB:CSOC:JOC and WB:GOC:JOC in molybdate method of Heinonen and Lahti (1981) with slight 2:1:1 ratio). Various substrates i.e. wheat bran (WB), modification. In the present study, 25 l of the enzyme Neem oil cake (NOC), cotton seed oil cake (CSOC), solution was added in 525 l of acetate buffer, which was groundnut oil cake (GOC) and Jatropha oil cake (JOC) pre-incubated at 37°C. After that, solution was further were taken separately and prepared by autoclaving 25g incubated for another 30 min at 370C temperature. Optical of each substrate in 250 ml of distilled water at 1210C for density of the solution was measured at 400 nm by adding 60 min, cooled and subjected to squeezing through the 4 ml of colour reagent. One unit of phytase activity was muslin cloth. For the development of microbial culture, defined as the amount of enzyme required to liberate 1 M method of Kim et al. (1998) was used in the study. The of phosphate per minute under the assay condition. extracts obtained from different substrate combinations were used as carbon and nitrogen sources for the growth Protein estimation of microbe. For this purpose, (NH4)2 SO4, 0.04g;

MgSO4.7H2O, 0.02g; KH2PO4, 0.05g; K2HPO4, 0.04g were added in 50 ml of substrate extract by adjusting the pH The estimation of protein content in different samples was to 6. The final volume was made upto 100ml with the carried out by using the method of Lowry et al. (1951) same substrate extract. It was then autoclaved and with bovine serum albumin as standard. allowed to cool and further used for phytase production

26 Purification of crude extract Table 1. Data on Biosynthesis of phytase (U/ml) from Pseudomonas sp. using ten different substrate combinations Initial purification was achieved by mixing the crude enzyme with the same amount of pre chilled (at -200C) Substrate Ratio Phytase acetone. It was mixed properly and the resulting precipitate combination activity* (U/ml) was allowed to settle for 1h. The clear upper layer of the WB+NOC 1:1 1.102 solution was siphoned off and remaining solution was WB+CSOC 1:1 0.305 removed by centrifugation at 10000 rpm for 15 min. The WB+GOC 1:1 0.527 pellets were dissolved in the 0.5M sodium acetate buffer WB+JOC 1:1 0.566 (pH 5) centrifuged at 10000 rpm for 10 min. and finally the WB+NOC+CSOC 2:1:1 0.396 supernatant was taken into another tube and stored at WB+NOC+GOC 2:1:1 0.436 0 4 C until further use. The final purification was done by WB+NOC+JOC 2:1:1 0.511 gel filtration using Sephadex G-50 followed by Ion WB+CSOC+GOC 2:1:1 0.572 exchange chromatography using DEAE-cellulose ion WB+CSOC+JOC 2:1:1 0.748 exchange material. WB+GOC+JOC 2:1:1 0.758 *Values are average of triplicates Characterization of phytase enzyme 0.511, 0.572, 0.748 and 0.758 U/ml respectively from WB+NOC, WB + CSOC, WB + GOC, WB + JOC, WB + Characterization of phytase determining the molecular NOC + CSOC, WB + NOC + GOC, WB + NOC + JOC, weight was done by SDS-Polyacrylamide Gel WB + CSOC + GOC, WB + CSOC + JOC and WB + Electrophoresis method adopted by Laemmli (1970) with GOC + JOC (Fig. 1). Literature survey also revealed that the slight modifications. work carried out using different oil cakes and wheat bran for the production of phytase and reported the similar trends in the phytase yield as obtained in the present Results and Discussion investigation.

Isolation and identification of phytase producing microorganisms from soil, in situ Optimization of incubation period

The isolate was found to be Gram negative, rod shaped Based on the findings of above experiment, the best and aerobic which showed similarity to the group-VIII substrate combination of WB+NOC (1:1 ratio) was used mentioned in Bergey's Manual. For the confirmation of for further studies at varied incubation periods for phytase isolates, different tests like catalase test, motility and production using Pseudomonas sp. (Fig. 1). The results endospore test were performed. The findings showed that obtained revealed the values of phytase activity as 1.003, isolated bacteria from ashvagandha-nilgiri field was found 1.029, 1.102, 1.069 and 1.049 U/ml respectively at to be gram negative, rod shaped under aerobic growth incubation periods of 44, 46, 48, 50 and 52 h (Table 2). with negative endospore test, positive catalase and positive These observations showed that the highest phytase motility tests confirming to the Pseudomonas sp. Similar activity was recorded at 48 h of incubation period. strain has also seen isolated from soil by Sri-akkharin et WB + NOC WB + CSOC WB + GOC al. (2006) and identified as P. fluorescence (PH02) by WB + JOC WB + NOC + CSOC WB + NOC + GOC WB + NOC + JOC WB + CSOC + GOC WB + CSOC + JOC Gram staining and colony morphology. WB + GOC + JOC 1.2

1 Biosynthesis of phytase (U/ml) from Pseudomonas sp.

0.8

Data on the values of phytase activity from Pseudomonas 0.6 sp. using ten different substrate combination are presented

PhytaseActivity (U/ml) 0.4 in Table 1. Among the substrate combinations, WB+NOC (1:1 ratio) was found to be the best combination for 0.2 maximum production of phytase i.e. 1.102 U/ml. The 0 phytase activity (U/ml) values for different combinations Substrate combination Fig.1 Biosynthesis of phytase (U/ml) from Pseudomonas sp. in ten substrate Combinations. were recorded as 1.102, 0.305, 0.527, 0.566, 0.396, 0.436,

27 of 34.48% from 4.64 fold purified phytase. The molecular mass of the purified phytase obtained from Pseudomonas

Table 3. Purification of phytase produced from Pseuduomonas sp.

Purification stages Total Total Total Specific Recovery Purification protein activity activity (%) folds (mg) (units) (U/mg of protein) 18.9 11.02 0.583 100 1 8.3 6.18 0.744 56.07 1.27 3.7 4.89 1.32 44.37 2.26 Ion Exchange chromatography (DEAE-Cellulose) 10.0 1.4 3.8 2.71 34.48 4.64 Biotechol 7(2):6-11

Where T 1=reaction time (in sec.) before drug administration, T2=reaction time (in sec.) after drug administration The model of experiment on inflammation was

Group I - Aqueous extract of Noni (250 mg/kg bwt. orally) Group II - Aqueous extract of Noni (500 mg/kg bwt. orally) Group III - Aqueous extract of Noni (1000 mg/kg bwt. orally) Group IV - Alcoholic extract of Noni (250 mg/kg bwt. orally) Group V - Alcoholic extract of Noni (500 mg/kg bwt. orally) Group VI - Alcoholic extract of Noni (1000 mg/kg bwt. orally) Table 1. Analgesic activity of orally administered aqueous and alcoholic extracts of Noni by hot plate method

Treatment Dose Before (mg/kg) drug oral Aqu. ext.of Noni 250 3.26a±0.17 Aqu. ext.of Noni 500 3.08a±0.24 Aqu. ext.of Noni 1000 3.14a± 0.11 Alc. ext.of Noni 250 3.15a± 0.37 Alc.ext.of Noni 500 3.22a± 0.13 Alc ext.of Noni 1000 3.28a± 0.17 Each treatment consists of six rats Means in a particular class (row) with different superscripts differ significantly from each other Means of a particular class (row) with at least one alphabet as common superscripts do not differ significantly from each other

post-mortem cases showing lesions of visceral gout, nephritis and bronchitis. The clinical signs noticed were deposition of urates on kidneys, heart and other organs. These organs were collected in a sterile manner and washed thoroughly in sterile PBS. The organs were processed by homogenization. Equal volume of chilled PBS was added to the homogenate to make 50% suspension. Processing was done in 3 repeated steps i.e. freezing-thawing for 3 times after 10 min interval. Then the homogenate was centrifuged at 7500rpm for 20 min and the supernatant was collected and filtered through 0.45µm syringe filter. The supernatant collected was stored at -20°C and used for detection of IB through ELISA. costly. In present study we explore the effectiveness of ELISA for post-mortem samples. Two rabbits were immunized for IB using vaccine strain for the production of anti-IBV antibodies. The presence of IB antibodies was demonstrated by western blotting assay. Recombinant N protein showed reaction with rabbit anti-IBV sera collected from hyperimmunized rabbits. The 52 and 45 kDa bands were predominantly seen on blot using rabbit anti-IBV sera as primary antibody in WB assay (Fig. 1). Indirect ELISA becomes a routine method for easy diagnosis because uses of anti-species antibodies make it convenient to use same tagged secondary vaccine sample OD sample OD ratio Jabalpur (MP) 1.079 16.3 Namakal (TN) 1.334 20.2 Jabalpur (MP) 1.336 20.2 Bilaspur (CG) 1.245 18.9 Durg (CG) 1.299 19.7 Rajnandgaon (CG) 1.349 20.4 Anand (GJ) 0.663 10.1 Anand (GJ) 1.159 17.6

Blank 0.035 Negative control 0.066 8192 0.091 was kept 25 kg/ha in both the crops in T7 and T1. The wheat variety (durum) was HD 4672. The wheat crops was sown in last week of November in all the years. All the recommended package of practices were adopted in both the crops.

Results and Discussion

Plant height of rice was maximum (114.44 cm) under the application of 100% NPK through fertilizer and 25 kg zinc sulphate/ha (T7) followed by 103.21 cm under 50% NPK given through fertilizer and 50% N through FYM in T1 (Table 1). Height was significantly lower in all the organic farming T5-T2+Rock phosphate+PSB+Azopirilium

T6-T2+Azospirilium+PSB

T7-100% NPK through fertilizer+ZnSo4 @ 25 kg/ha as per soil test (120 kg N, 60 kg P2O5 and 40 kg K2O/ha)

T8-Dummy Plot as T2

SEm± CD at 5% 4.09 10.10 14.15 6.10

*figures in parentheses are % yield over T7 T5-T2+Rock phosphate+PSB+Azopirilium

T6-T2+Azospirilium+PSB

T7-100% NPK through fertilizer+ZnSo4 @ 25 kg/ha as per soil test (120 kg N, 60 kg P2O5 and 40 kg K2O/ha)

T8-Dummy Plot as T2

SEm± CD at 5%

*figures in parentheses are % yield over T7 (55.62%)* 42.34 59276 16226 1.37 (64.32%)* 38.71 54194 6344 1.13 (58.81%)* 37.74 52836 30316 2.34 (57.33%)* 39.93 55902 12732 1.29 (60.66%)* 65.82 78984 53934 3.15 (0.00%)* 40.97 57358 14308 1.33 (62.24%)* 1.48 CD at 5% 4.49

and T10, 120 kg N + 60 kg P2O5 + 40 kg K2O + 5 t FYM + 500 g Azotobactor/ha. The doses of SSNM treatment were workedout on the basis of equation developed by Soil Test Crop Response for rice and wheat crop. These treatments were applied to both rice and wheat crop every year. The available N, P, K, S, Zn, B and Mo content of the soil was 265, 9.10, 298, 9.20 and 1.4 kg/ha respectively. Treatments were kept in randomized block design with four replication and plot size of 33.6 square meters. Crop varieties were 'NPT 24-36' and 'HI-4681' for rice and wheat, respectively. Rice and wheat seeds were treated with bavestin @ 2.5 g/kg seed and sown in 20 cm apart rows in three consecutive years in the same fields and treatments were allocated as per the plan and layout. lowest seed yield of 4.38 t/ha with recommended dose of fertilizer . This indicate that escaping of essential elements under T8 and T4, T5, T6 and T7 ensure the production of poor yield attributes as compared to T1 and T9 which ultimately resulted in reduced grain yield.

Equivalent yield

The significantly highest wheat equivalent yield was noted under T9-120 kg N+60 kg P2O5 + 40 kg K2O + 10 t FYM/ha (11.20 t/ha/year) applied to both crop components and it was closely followed by T1-150 kg N + 90 kg P2O5 + 60 kg K2O + 10 kg S + 5 kg Zn + 1.5 kg B + 0.5 kg Mo/ha components along with 10 t FYM/ha applied to rice closely followed by T10 where state recommended fertilizer were applied to both crop with Azotobacter seed inoculation along with 5 t FYM/ha applied to rice. The lowest nutrients availability after harvest of the 2006-07 crop cycle, was noted in T8 where state recommended fertilizers (120:60:40 kg/ha) were applied. These results are in close conformity with the finding of Das et al. (2003). The present study indicates that integrated nutrient management as application of 120 kg N + 60 kg

P2O5 + 40 kg K2O + 10 t FYM/ha (T9) produced maximum grain and straw yields of both rice and wheat crop which were comparable to those obtained with 150 kg N + 90

Results and Discussion

Effect of nitrogen

The observations in relation to leaf area index, root dry weight, dry matter accumulation per plant at different stages of crop growth as well as green and dry matter yields were recorded at 50% flowering (harvesting) stage exhibited architectural differences amongst the various treatments. Levels of N significantly increased the LAI at all the stages of crop growth except at tillering (30 DAS) during first year (Table 1). The increasing level of nitrogen from 40 to 100 kg N/ha could not exhibit marked variations well as in pooled data respectively.

Effect of supplemental sources

Supplemented sources also had significant effect on this parameter at all the stages during both the years except at tillering in 2003-04. The supplemental sources of vermicompost superimposed with Azotobacter secured higher values of 3.05, 3.31, 5.55, 5.63, 7.89, 8.71, 7.54 and 7.50 at 30, 60, 90 and 50% flowering stages during both the years, respectively, and also proved significantly superior over FYM alone with respect to stages as well as the years. Rawat CR, Hazra CR (1993) Effect of cutting management practices and N application to promising varieties of oat. Annual Report IGFRI Jhansi p 155 Sivasankar A, Bansal KC, Abrol YP, (1993) Nitrogen in relation to leaf area development and photosynthesis. Proc Indian Natn Sci Acad B 59:235-244 Vyas MN, Ahlawat RPS, Patel JS, Baldha NM, Malavia DD (1988) Response of forage oats to varying levels of nitrogen and phosphorus. Indian J Agron 33:204-205

(1:50 ratio) was applied in furrows prior to sowing. The nitrogen was applied in the form of urea in three split equal doses, first at sowing time as a basal dose (1/3 dose), first top dressing at 20 days after sowing (1/3 dose) and second top dressing at first harvest (1/3 dose). The intercultural operations were performed by hand wheel hoe at 20 DAS and first hand weeding at 30 days after sowing and second hand weeding after first harvest. The data pertaining to biometrical observation as well as green fodder and dry matter yield were recorded at first harvest and second harvest of the crop as per the treatments. 1.25 0.23 44 10 107 24 2.89 0.27 80 19 186 42 3.06 0.28 87 20 208 47 4.49 0.31 116 27 273 61 4.67 0.32 130 30 299 68 6.48 0.35 149 35 342 81 6.65 0.39 155 36 378 88 0.02 0.05 1.53 0.36 2.84 2.07 0.07 0.014 4.62 1.10 8.55 6.24

4.02 0.31 104 24 233 53 3.63 0.28 97 22 239 55 0.01 0.03 1.50 0.03 0.98 2.82 CD at 5% 2.11 NS 0.11 33.66 0.03 0.07 NS 0.08 2.96 NS 334 350 81 82 377 380 88 89 2.83 2.83 2.07 2.07 8.55 8.55 6.24 6.24 4.01 4.01 2.93 2.93 NS NS NS NS 4.01 4.01 2.93 2.93 NS NS NS NS 1.72 1.72 3.00 3.00 NS NS NS NS 3.48 3.48 6.01 6.01 NS NS NS NS 2.79 2.79 7.97 7.97 CD at 5% NS NS NS NS of nitrogen on forage sorghum varieties. MSc(Ag) Thesis Gujrat Agriculture University Navsari Chemical analysis

The ash content, oil content and protein content in the sample was estimated according to AOAC (1980). Total carbohydrate was estimated by the acid hydrolysis process (Hassid and Abraham 1973). Calcium and Magnesium in the above aliquot were determined by the Versenate titration method (Black 1965). Phosphorus content in the Diacid digested aliquot was determined by Vanado-Molybdo Phosphoric Acid method (Koenig and Johnson 1942). in extrudates of blend-2 consisting of rice:soybean:potato- 80:10:10 and lowest (6.85%) in extrudates of blend-1 (Rice: Soybean: Potato-100:0:0). There were no specific trends of variation in protein content of extrudates as influenced either by moisture level or by barrel temperature. These results are in agreement with the findings of previous investigators Singh (2006b); Obatolu et al. (2005); Berrios et al. (2004). Addition of soybean at the level of 10% increased the level of protein as reported by several investigators Singh (2006a); Adesina et al. (1998); Iwe and Ngoddy (1998); Iwe (1998). (2005); Berrios et al. (2004). Blend-1 having 100% rice contained negligible amount of oil content (0.42- 0.68%). Inclusion of soybean at the level of 10% in the blend-2 and blend-3 increased the oil content of product because soybean is rich in oil. Similar results have been also observed by Iwe (1998); Singh et al. (2003).

Ash content of extrudate

The ash percentage ranged in between 0.84 to 1.68%. Highest ash content (1.68%) was found in extrudates of blend-3 (rice: soybean: potato-70:10:20) and lowest the phosphorus was in the extrudates made from blend-2. There were no effect on various minerals viz., calcium, magnesium and phosphorus due to change in feed moisture and barrel temperature. These results are in same pattern as reported by Singh et al. (2006); Obatolu et al. (2005); Berrios et al. (2004). It is evident that inclusion of soybean and potato in rice blend increase minerals viz., calcium, magnesium and phosphorus of extrudates similar supported by Singh et al. (2003); Iwe (1998).

mushrooms, acting together to hydrolyse cellulose. Due to these enzymes mushroom bioconvert in edible plant waste into directly palatable food. Enzymatic browning is a chemical process which occurs in fruits and vegetables by the enzyme polyphenol oxidase which results in brown pigments. They are a class of enzymes that were first discovered in mushrooms and are responsible for deterioration. They are a group of copper proteins that are widely distributed from bacteria to mammals (Roy and Bahl 1984). They are also referred to as phenol oxidase, phenolase, monophenol oxidase, diphenol oxidase and tyrosinase. T1 (0.20) 101.00

T2 (0.40) 131.67

T3 (0.60) 160.34

T4 (0.80) 205.34 SEM 0.98 CD at 5% 2.20 H. ulmarius

T0 (Control) 70.00

T1 (0.20) 114.34

T2 (0.40) 158.00

T3 (0.60) 202.67

T4 (0.80) 256.67 SEM 1.54 CD at 5% 3.45 0.83 256.67 g/30 min/ml

10 0.0238

(Hostathion, Bayer Crop Science, Ltd., Gujarat) on the growth and sporulation of Sclerotium rolfsii The test was conducted using poisoned food technique . Concentrations were worked out on the basis of formulated product.

In vitro efficacy of herbicides

Seven recommended herbicides viz., Imazethapyr (Pursuit 10 SL, BASF (I) Ltd.), Pendimethalin (Eraser 30% EC, Jai Shree Rashayen Udyog Ltd.), Glyphosate (Shriram finding reported by Rathod and Patel (2003).

In vitro efficacy of leaf extract

Out of eight botanicals (Table 4) neem was found less effective at 10 per cent concentration, but found inhibitory at 20 and 30 per cent concentrations against Sclerotium rolfsii. Similar finding have been reported by Hanumanthegowda (1999); Ume et al. (2001); Kolte and Raut (2007); Singh et al. (2007); Suryawanshi et al. (2007); Mandhare and Suryawanshi (2008). Complete inhibition (100%) was recorded only in nilgiri at 30 per cent concentration conforming the finding of Suryawanshi 88 1.90x1.88 0.91x0.88 43 1.77x1.76 0.88x0.76 - - -

------448 2.01x1.96 0.80x0.73

CD at 5% 1.54 *Mean of three replications value in parenthesis are transformed values 00 - - 00 - - 00 - -

38 1.98x1.93 0.97x0.93 00 - - 00 - - 448 2.01x1.96 0.80x0.73

CD at 5% 2.75 *Mean of three replications value in parenthesis are transformed values 2,4-

2,4- 357 1.94x1.89 0.92x0.86 275 1.92x1.85 0.90x0.82 221 1.91x1.85 0.89x0.81

278 1.85x1.78 0.93x0.89 259 1.83x1.77 0.91x0.84 220 1.81x1.76 0.89x0.83 448 2.01x1.96 0.80x0.73

CD at 5% 3.24 *Mean of three replications value in parenthesis are transformed values Jinantana K (1985) Studies on damping-off of castor bean seedling caused by Sclerotium rolfsii Sacc. Bangkok (Thailand) 118 leaves Jinantana K, Tharmmasak S (1986) Effectiveness of fungicides against castor seedling blight (Sclerotium rolfsii) Sacc. J Thai Phytophalogical Society (Thiland) 5(1):25-32 Johnson M, Reddy PN, Reddy DR (2008) Comparative efficacy of rhizosphere mycoflora, fungicides, insecticides and herbicides against groundnut stem rot caused by Sclerotium rolfsii. Ann Pl Prot Sci 16(2):414-418 Kaur A, Gupta VP (2003) Evaluation of seed dressing fungicides for management of Sclerotium collar rot in lentil. Indian J Pulse Res 16(1):75-76 towel-paper method. The percentage of germination is tested after seven days, which depends upon the number of seeds germinated. At the field level again fixed number of seeds is grown in the field and their percentage is calculated after few days. At present there are no instrumental methods for testing the germination percentage even at lower level of accuracy. In seed industry, before marketing the seed it is necessary to determine percentage of every seed lot for quality control. The present method of determination of germination percentage of seed samples in laboratories is being done in germinator where about 10-15 days time is normally taken. The method is the standard method samples having poor germination (30%) were having conductivity more than 1.0 mS (milisiemens), whereas, samples having more than 70% germination gave conductivity less than 0.6 mS after 4 hr interval. It can be broadly concluded that samples having conductivity less than 0.6 mS after 4 hr will have more than 70% germination. If the samples of the same variety are collected from the different sources and conductivity measurements are performed than the user can select best quality out of available lots which give less conductivity after 4 hour. The conductivity also slightly varies from variety to variety for the same germination level. However, it further needs exhaustive study how it varies from variety to variety. 0.222 0.344 0.913 0.177 0.275 0.733 0.227 0.351 0.897 0.177 0.282 0.716

46% 86% 0.273 0.389 0.897 0.142 0.234 0.502 0.281 0.362 0.881 0.147 0.242 0.523 0.271 0.374 0.913 0.141 0.245 0.521 0.275 0.375 0.897 0.143 0.24 0.515

58% 84% 0.189 0.331 0.789 0.156 0.255 0.534 0.198 0.326 0.794 0.167 0.263 0.541 0.195 0.33 0.781 0.162 0.247 0.548 Average 0.194 0.329 0.788 0.162 0.255 0.541 Ltd New Delhi Krishna Kant (2010) Microprocessor-Based Agri Instrumentation. PHI Learning Pvt Ltd New Delhi information under such complicated system is unpredictable. Therefore models developed for various crops for prediction of crop yields, forecasting of on-set of disease and pest infestation have not served much purpose. Keeping this in view, it is envisaged to incorporate the presently available sustainable agricultural technologies of tillage, biodiversity, use of bio-fertilizer, integrated nutrient management, integrated paste management of 20 crops. All types of variations in technologies due to agro- climatic reasons will also be incorporated. Economic and field information of the user is necessary to provide right type of information by the software and after obtaining A

Different Field Sowing time Varieties Preparation & method

Production Post harvest Marketing technologies technology strategies

Stop Nand K Fageria Growth and Mineral Nutrition of field Crops, Taylor and Francis publication. Thind TS Diseases of Field crops and Their Management. Daya Publishing House New Delhi breed from home tracks viz., Khargone and Badwani districts of MP and Government cattle breeding farms. The DNA was isolated by the method as described by John et al. (1991). Quality check and quantification was done by Nanodrop TM spectrophotometer and electrophoresis on 0.8% agarose gel. The DNA concentration was determined and samples were diluted 10-30 times (approx. 30 ng/µl) with MiliQ water.

Amplification of BoLA-DRB3.2 gene

BoLA-DRB3.2 gene was amplified from genomic DNA samples by polymerase chain reaction. The HL-030 and I 0.080 X 0.113 II 0.032 XI 0.016 III 0.048 XII 0.032 IV 0.032 XIII 0.016 V 0.064 XIV 0.032 VI 0.032 XV 0.016 VII 0.080 XVI 0.032 VIII 0.032 XVII 0.129 IX 0.063 XVIII 0.0145

Acknowledgements The work has been carried out under the project "Molecular characterization of four breeds cattle found in Madhya Pradesh" sponsored by Department of Biotechnology, New Delhi for which we are thankful to DBT.

to conduct research to study the information seeking behaviour and constraints in effective dissemination of agricultural information, with the following objectives: • To know information seeking behaviour of extension personnel • To know the association between profile of extension personnel with their information seeking behaviour • To know the constraints perceived by extension personnel and suggestions for effective dissemination of agricultural information The results of this study will help to know the sources and methods used by agriculture extension aspiration level, 10.00 per cent had low information seeking behaviour, 50 per cent had medium and 40 per cent had high information seeking behaviour. In case of out of total 34 extension personnel 29.41 per cent had low, 50 per cent had medium and 20.59 per cent had high information seeking behaviour (Table 3). Similarly, out of total 26 extension personnel who had high aspiration, 34.63 per cent had low information seeking behaviour, 23.07 per cent had medium and 42.30 per cent had high information seeking behaviour regarding improved agricultural technology. The chi square value 6.568 was found to be significant at 0.05 level of probability. Therefore it can be (46.25%), appropriate and number of agricultural literature should be provide to the extension personnel (26.25%),

Table 5. Constraints perceived by the extension personnel in effective dissemination of agricultural information

Constraints Frequency Percentage 59 73.75 23 28.75 48 60.00 37 46.25 19 23.75 Lack of awareness in extension programme among the farmers 13 16.25 Photocopying, printing, photography and transport facilities are not available 33 41.25 Agricultural information is not available timely 26 32.50 References

Hakimi H (1997) A study of information-seeking behaviour of researchers at the Iranian Natural Resources Research Centre. MSc(Ag) (Thesis) University of Tehran Tehran Iran

farmers, 100 extension personnel's and 50 agro-input providers were selected from 217 villages (Table 1). Selection of villages was based on availability of accessing mobile network. Two SMS in a week i.e. Tuesday and Friday are issued on various agricultural developments like weather forecast, disease forecast, availability of input, critical stages, Plant protection measure, Livestock production and management, market information. Kisan Mobile Sandesh is also being used as a medium to send information on important trainings and other programmes to the contact farmers of KVK. All selected KMS users who are having their own cellular mobile handsets supporting all major GSM/CDMA networks within the study area can receive the SMSs from KVK Chhindwara and Block- Seoni 60 5 Block-Kurai 40 5 Block-Barghat 45 5 Block-Chapara 50 5 Block-Lakhanadon 45 5 Total 510 50 Approach area of one agro-input provider is 5 to 25 villages

Results and Discussion

To know the impact of Kisan Mobile Sandesh the content of evaluation was studied in ten dimension viz., receive need based information, spend few seconds for receive information, easy to understand, appropriate time of Source of information-KMS Extn. Agro-input Mean Other personne providers source of l's (25) information (50) (167) 47 24 163 62 50 25 167 22 50 25 146 84 50 25 167 77 50 24 138 30 42 22 132 23 44 16 124 41 40 18 136 28 27 15 81 22 Strong linkage with KVK 92 50 24 166 08 technique in hypsometric analysis of digitized contour maps helps in improving the accuracy of the results and saves time. Considering the above points, a study was undertaken to determine the geological stage of watershed development for subwatersheds of Kanhiya Nala watershed located in Satna and Panna district of Madhya Pradesh.

Material and methods

The study area Kanhiya Nala watershed which lies within the Tons river catchment 80032'24'' to 80034'12'' E longitude and 24006' to 24010'48'' N latitude (Fig 1) with elevation The area below the respective curves is measured and its proportion with reference to the total area of the graph calculated, which is known as the hypsometric integral (HI). Hypsometric integral value for each of the watershed was determined (Table 2). The values of hypsometric integrals and the form of the hypsometric curves taken together help to identify the stage of basin development (Strahler 1952). The threshold limits recommended by Miller (1964). As given below were adopted for deciding the stage of watershed. (1) The watershed is at inequilibrium (youthful) stage if the HI 0.6. The HI values for all the subwatersheds ranged from 0.39 to 0.67. None of the watershed is at monadnock stage. The subwatershed number 1, 2, 3, 4, 5 and 8 are at equilibrium stage, which is evident from the HI values of 0.39 to 0.56 (Table 2). The subwatershed number 6 and 7 are at youthful stage, which requires soil conservation measures as they are likely to experience further erosion before coming to ultimate stage of the erosion cycle. Hypsometric analysis is useful to comprehend the erosion status of a watershed and prioritize them for undertaking soil conservation measures. The analysis can be performed with easily extractable information from to the solvent extraction plant for oil extraction. Several methods of stabilization of bran by enzyme inactivation involving heat treatment, dehydration, steam treatment, heat extrusion and even cold storage have been suggested. But temperature range is critical. Research works are in progress on the development of simple technologies for the stabilization of rice bran obtained soon after the polishing. Different units have been developed for this purpose working on the principle of hydrothermal treatment (Chakravarty 1981), conduction heating (Patel 1982), fluidized bed type (Khem Chand 1974) radiation heating by infrared lamps (Narain et al. 1982), extrusion with higher friction (Saunders 1984). But placed in a soxhlet apparatus. The apparatus was fitted with condenser and flask containing sufficient petrol to work as the extractor. Heat the flask on a sand bath placed on an electric heater for about 6 hours. The extraction was complete when drop of petrol taken from the dripping of the extractor leaves no greasy state on a filtler paper. Remove the thumble and heat so that extractor was filled about 2-3rd with petrol and only a small quantity of petrol is left in the flask. The boiling was stopped at this point and the flask was disconnected. The residual petrol was filtered (containing fat in solution) through filter paper and was collected the filter in a weighed 100 ml beaker or a glass dish. The flask and filter paper was washed a no. of times with redistilled petrol till a Results and Discussion

Effects of various treatments on shelf life of bran

The rice bran was subjected to various treatments selected under the study, such as vacuum and nitrogen treatment. The results obtained in terms of FFA content during storage were analyzed and presented in this section. As the rice bran has been reported to become useless beyond 10% FFA content (Chakrawarty 1995), this limit was selected as the index for determination of shelf life. vacuumized condition the FFA of rice bran remained within safe limits up to 2 months of storage duration.

Treatment SS MSS DF F 1 8.0408 0.58316 x 10-2 1 0.1835 1 4.7836 1 522.0311 1 4.14636 Error 0.921726 0.31784 x 10-1 29

CV= 0.267126, Std = 0.006654, Se Diff. = 009411, t value =2.085964CD=0.019630

effectively combined into snacks of high nutritive value, which is regularly consumed during teatime and breakfast. This product is expected to be good source of protein and minerals and will provide sustainable energy to school going children and pregnant ladies while serving as a popular snack.

Material and methods

Sorghum, Horse gram and Defatted soy flour were procured from local market of Jabalpur. The procured sorghum and horse gram were thoroughly cleaned and all the impurities including broken were removed and -3 -3 X4 + 9.50 x 10 X1X2 - 6.875 x 10 X1X3 - 0.017 X1X4 + -3 -4 2 0.012 X2X3 + 8.75 x 10 X2X4 + 1.25 x 10 X3X4 - 0.101 X1 2 2 2 - 0.127 X2 - 0.073 X3 - 0.073 X4 ...... 1 From the tabulated values, three-dimensional graphs were prepared treating two independent variables

Code levels Parameter -2 -1 0 1 2 12 15 18 21 80:10:10 75:15:10 70:20:10 65:25:10 60:30:10 125 130 135 140

Screw speed (X4), rpm 120 125 130 135 140

researchers and research systems everywhere are witnessing major changes in the ways in which the results of research, and the ensuing applications and impacts, are being made accessible and communicated, the processes by which knowledge, information and data are generated and shared are being transformed and reinvented. According to Linggawati (2002), now with the fast growth of information, it is almost impossible for any libraries to own complete Information. A library has to build the networks to have wider information access to fulfill the complexity of the users' needs. Witten and David (2003), defined digitization as the process of taking traditional library materials that are in form of books and papers and converting them to the electronic form where With the invention of modern information technology and development of digital storage media it has become practically possible to store content of printed holdings in digital format. Hard disc installed in computers have emerged as gigantic inventory of digital storage. Virtually limitless amount of digitized information can be stored in such media. A huge amount of voluminous data can be stored in a single disc or drive. The basic aim of e- preservation is to convert the printed holding into digital format. Such conversion is possible through scanning procedure. The material can be scanned in editable and backups must be taken, quality enhancing and loss prevention softwares are advisable to be used. Durability - It is the feature due to which e-preserved documents are preferred, e-preserved documents are found to have more life then physical form of holdings/ documents. Durable media provides us with a facility of data loss prevention and unauthorized adulteration of digitized holdings. Emulation - It is an approach which gives the e-preserved holdings a newer dimension, with invent of computers we have witnessed great changes in performance, design, specifications and in utility of computers. Now we have document should be so secure that no; alteration, modification, mutilation or format change should be possible at any stage of the process and its use. Apart from this the e-preserved accessible files must be free from viruses, spywares, malwares etc, so that data losses are prevented Skill - To convert printed information into electronic/digital format great skills are required since the information has already been compiled, tabulated, classified, accessioned and stored in Library. Multi dimensional application skills in soft-computing and library knowledge are essential carry out the task apart from maintaining the original materialistic identity and integrity of the document before and after e- • Shoddy storage, overuse and misuse of e-preserved holdings and not taking digital and analog backups.

• Lack of sufficient training to staff and to the users who access the facility of usage of e-preserved holdings.

• Skeptical and cynical nature of employees with resistance and willingness to adopt technology and good work practices and ethics.

• Unawareness of existing digital resources and facilities in the library. preservation is a technique which assists in providing wanting information in a single instance and in multiple desired formats. It is essential to adopt new technology get adapted to it, technological and conceptual changes need to be incorporated as soon as possible. A well designed framework of e-preservation of holdings needs to be implemented to secure holdings and provide users with optimum access to it. The important thing is integration of traditional and modern technology to promote library and information science activities and in turn to benefit mankind the information seekers. It is further expected that this paper will serve to provide a conceptual framework of issues and methodology for successful e- preservation of massive library holdings.

ISSN : 0021-3721 JNKVV Volume 44 Research Journal Number (1) 2010

Contents Research Paper Resin production system in Guggul plant [Commiphora wightii (Arnott) Bhandari] 1 Moni Thomas, Atul Shrivastava and S.S. Tomar

Upland rice in Madhya Pradesh: Problems and Prospective 9 I.M. Khan, R.K. Tiwari and S.K. Rao

Production, purification and characterization of phytase from Bacillus subtilis 19 Namrata Verma, Keerti Tantwai, L.P.S. Rajput, Sunil Kumar, Iti Gontia and Sharad Tiwari

Production of phytase from soil isolates of Pseudomonas sp. 25 using agricultural byproduct substrates Yuvraj Soni, Keerti Tantwai, L.P.S. Rajput, Sunil Kumar, Iti Gontia and Sharad Tiwari

Analgesic activity of aqueous and alcoholic extracts of Noni (Morinda citrifolia) 31 by hot plate method Bharti Jethani, R.K.Sharma and Sudhir Kumar Jain

Detection of infectious bronchitis virus in suspected post-mortem field tissue 35 samples by ELISA Sudhir Kumar Jain, Hemlata Jain, Megha Kadam Bedekar, Bikas Chandra Sarkhel and Sanjeev Singh

Integrated nutrient management in rice wheat cropping system 39 B.M. Maurya, Jyoti Dekate and V.B. Upadhyay

Evaluation of site specific nutrient management on productivity and 45 economics of rice-wheat crop sequence V.B. Upadhyay, S.K. Vishwakarma and Vikas Jain

Effect of inorganic, organic and sources of nitrogen on growth and yield of 49 fodder oat (Avena sativa L.) Anay Rawat and S.B. Agrawal

Response of sorghum [Sorghum bicolor (L.) Moench] cultivars to nitrogen and 53 phosphate solubilizing bacteria under different cutting intervals B.D. Ghode, Seema Bhadauria and S.D. Sawarkar Effects of inclusion of soybean and potato on nutritional and sensory quality of 57 rice based extrudates Rajendra Singh Thakur and A.K. Tomar

Extracellular enzyme profile of oyster mushrooms as affected by gamma radiation 63 Alpana Singh and Anshu Verma

Screening of pesticides against Sclerotium rolfsii causing collar rot of soybean 67 Ashish Kale and R.K. Varma

Microcontroller based seed germination testing device for soybean 75 A.K. Rai and Bharati Dass

Web enabled software for integrated farm production management of 79 major crops grown in central India for sustainable growth Bharati Dass and A.K. Rai

Molecular characterization of BoLA-DRB3.2 allele in Nimari breed of cattle by 83 single strand conformational polymorphism (SSCP) R. Ranjan, C.D. Bhong, K.N. Chavan, S.N.S. Parmar and C.G. Joshi

Information seeking behaviour of extension personnel regarding improved 87 agricultural technology N.K. Khare, A.K. Wankhade and Tapan Chandra Kar

ICT based Kisan Mobile Sandesh-an innovative approach 93 Nalin Khare and Abhay Wankhade

Hypsometric analysis of Kanhiya Nala watershed using geographical 97 information system S.K. Sharma, S. Tignath and U.C. Chaube

Effect of modified atmosphere storage (under vacuum and nitrogen) 101 on shelf life of rice bran S.P. Srivastava and A.L. Basediya

Effect of process and operational parameters of extrusion cooking of sorghum, 107 horse gram and defatted soy flour blends to produce protein rich ready-to-eat food snacks A.L. Basediya, S.P. Shrivastava and Sheela Pandey

An approach for possibilities for e-preservation of library holdings 113 Siddarth Nayak, Sharad K. Jain and P.S. Nayak

Issued 30th June, 2011