Genetic diversity of Frankia associated with Hippophae L. in Lachen Valley of North Sikkim

rrliesis Submitted for the Degree of Doctor of Philosophy in Science (Botany) under the University of North Bengal

Su6mittea 6y Bharat Chandra Basistha

Vnaer tfie supervision of Dr. Arnab Sen

Department of Botany University ofNorth Bengal Raja Rammohunpur, Darjeeling West Bengal, India 201 1 11\_, SeL·6~4D954Lb'7 ~ ~i4t

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This thesis is an outcome of years of research that has been carried out since I was enrolled as a Ph.D. student with the Department of Botany, University of North Bengal. During this time, I have worked with people of different hierarchy whose assorted contributions during my research and making of the thesis, deserve special mention. It is a pleasure to convey my gratitude to everyone in my humble acknowledgment. I must emphasize that I shall remain solely responsible for any unknowingly omissions and errors during this acknowledgement. I am heartily thankful to my supervisor Dr. Arnab Sen, Reader and Head, Department of Botany, University of North Bengal who undertook to act as my supervisor despite of his many other academic and professional commitments. His wisdom, knowledge, commitments, excellent guidance, valuable suggestions and constant encouragements to the highest standards inspired and motivated me. His scientific intuitions has made him oasis of ideas and passions in science, which exceptionally inspired and enriched my growth as a researcher. I am indebted to him more than he knows. This thesis would not have been completed without his valuable inputs. I sincerely express my deep gratitude and thank him. I express my gratitude to all the teachers of the Department of Botany, University of North Bengal for offering me necessary help during my research. My special thanks to Prof. A. P. Das, Department of Botany, University of North Bengal, for his involvement with his originality during writing my synopsis and at the end of the thesis too. I am grateful and express my thanks to the Department of Biotechnology, Government of India and Sikkim State Government for providing me project and the administrative support. I also express my sincere thanks to Shri. Bhim Dhungel, Hon'ble Minister, Department of Science & Technology, Government of Sikkim, for taking personal interest in encouraging me to complete my thesis. My gratitude is also to Shri. R. Telang lAS, Secretary, Department of Science & Technology, Government of Sikkim for supporting me by providing me ample leave to complete my work. I am indebted to Dr. Pushpa Tamang, Shri. D. G. Shrestha, Shri. D. T. Bhutia, Add!. Directors', Sikkim State Council of Science & Technology, for supporting me in different ways and handling my projects during my absence in the office. I also acknowledge Shri. D. P. Neopaney, Joint Director, Department of Science & Technology and his entire administrative team of the Department for their complete administrative support and timely process of documents related to my work. I also thank Smt. S. Pradhan, Sr. Accounts Officer, Shri. N. P. Sharma, Accounts Officer and the whole accounts section of the Department of Science & Technology and the Sikkim State Council of Science & Technology for accounts related works and timely releasing my PhD grant from the office. I am also thankful to Shri. S. R. Lepcha, Shri. K. B. Subba and Shri. Suman Thapa, all Asst. Scientific Officers' of Department of Science & Technology for their technical assistance from time to time. My special thanks to Prof. L. S. Tisa, Department of Microbiology, University of New Hampshire, USA and Dr. L. Rongsen Chinese Academy of Sciences, Chengdu Institute of Biology, Chengdu, China, for their personal feedback and suggestions. I acknowledge Prof. Philip Normand, University Clad Bernard Lyon I, Villeurbanne, Prof. D. D. Myrold Oregon State University USA, Prof. D. R. Benson Department of Molecular and Cell Biology, University of Connecticut, USA, Prof. I. G. Bartish, Balsgfird-Department of Horticultural Plant Breeding, Swedish University of Agricultural Sciences, Kristianstad, Sweden and Prof. L. G. Wall, Head Professor at UNQ, Science and Technology Department, Bernal, Argentina for sending me references and publications. I owe my gratefulness to Shri Pranaya Sharma (Poudyal), Kathmandu Nepal for collecting relevant publications for reference on my behalf, Shri. Sushen Pradhan, Junior Research Fellow, Shri. Narapati Sharma, Research Asst., Shri. Laydong Lepcha Data Entry Operator, Sikkim State Council of Science & Technology and S hri . lndra Mani 1\dhih.ari PS to the Mini~ter. Home Department. CJ m t. ol" Si h.h.i m who a ·sisted me in fi eld and laboratory wo r k~. I also thank, Shri Rajdeep Gurung Project Scienti!.l. Smt. Radha Basnet J RF, Ms. Srij ana Chelri JRr. Shri. Am Bahadur Driver. Sikki rn State Council of Science & Technology. Shri. Kewal Sharma and Shri Ram Bahadur Gurung Driver!.. Depanmem of Science & Technology, who had helped me in fie ld works and did cooking in the extreme climatic conditions too. My thanks are also to Late Sanbir Subba, S mt. Saroj Lepc.:ha and Smt. Dawa Gyalmo Bhutia, Lab Attendants' for caring and storing the germplasm collected from the field. l thank all the project fellows and staffs o f Sikkim State Council of Science & T echnology who are directly and indirectly involved during the cour e of my PhD work. I owe collecti ve and individual acknowledgements to my lab mates Dr. Balwinder Singh Bajwa, Dr. Manprit Gill, Shri . Debadin Bose, Shri. Arvi nd Goyal, Ms. Subarna Thakur, Ms. Ri tu Rai and Shri. Maloy Ghosh, whose pre ence was perpetually refre hing, helpful , memorable and providi ng me uch a pleasant time when working together. lt wi ll be remi s. if I don' t acknowledge Shri. Krishanu Data Entry Operator. Bioinformatics Infrastructure Facility and Khurhid Alam our Lab Boy. Many thanks go to Shri. Bikram Saha, Research Fe ll ow and my hostel mate for his self less devotion by helping me in carryi ng out PCR works. 1 owe thanks to my hostel mates Shri. Anand Sharma, Shri. Ritesh B iswa, S hri Saurav Moktan, Shri . Indra Karki and Shri Bhakta Kunwar for dry humour about scientist's life. Shri Bishwanath Gan, Shri. Rishikesh Mandai, Shri. Binay Patel, Shri. Diwakar Chaudhary, Shri . Anurag Chaudhary and Shri. Kapil Mitra fo r timely meeti ngs and creating such a great friendship and support when required. T hanks to my hostel cooks Shri. Mukund Roy (Robert), Shri. Prafulla Singh and Shri . Sushi! Chandra Burman for their regular delicacies during the stay at the University Research Scholars' HosteL I don' t know where I would be without my family. I owe my deepest gratitude, love and respects to my parents who deserve special mention for their inseparable support, blessings and prayers. My father Shri. U. C. Vasistha in the first place is the person who inculcated the fundamentals of learning, showing me the joy of intellectual pursuit ever since I was a child. M y mother, Smt. S. K. Vasistha is the one who unconditionally and sincerely rai sed me with her warm, caring and gently love. I also thank Kamala Sharma-Shri R. P. Sharma and Samjhana Dhakal-Shri Pramod Dhaka! my sisters and their husbands fo r constant encouragements. My sincere thanks to my younger brother Satish Basistha, his wife Bidhya Ratna Sharma Basistha and daughter Khushmana fo r cooperations, support and taking care of certain fa mily affairs in my absence. I thank the entire Basistha family fo r constant support and encouragement to complete my work. I don' t have words to express my appreciation to my better half Shobha Sharma Basistha whose sacrifice, dedication, love, persistent confidence in me and without complaints on household mauers has taken the load o ff my shoulder. 1 owe her for being unselfi shly let her intell igence, passions and ambitions mix with mine. Therefore, I also thank Shri D. D. Gautam and Smt. M. Sharma Gautam's family fo r letti ng me take her hands in marriage and accepting me as one of their famil y member, warmly. My thanks to my daughter Neelanjana a nd son Pratyush for being nice. not so demanding and disciplined children when I was in the university. Alo ng with thanks. I also apologize to you both for not being with you when I was needed sometimes. Above all , it may be incomplete acknowledgement if I do not thank god, without his blessings no work can be done, I believe. Finally, I thank each and everyone who were directly and indirectly imponant in successfully completing my thesis, as well as 1 express my apology to all whose name could not be mentioned

(Bharat Chandra Basistha) Contents

CHAPTER-I 1.0.0 INTRODUCTION CHAPTER-II 2.0.0 REVIEW OF UTERATURE 17 2.1.0 HISTORY OF HIPPOPHAE L (SEABUCKTHORN) RESEARCH 17 2.1.1 Legends associated with sea buckthorn 18 2.2.0 ANTIOXIDANT PROPERTIES AND CHEMISTRY 18 2.3.0 AGROTECHNIQUES OF SEA BUCKTHORN 20 2.4.0 DISTRIBUTION OF ACTINORHIZAL PLANTS 22 2.5.0 TAXONOMY AND EVOLUTION OF ACTINORHIZAL PLANTS 24 2.6.0 HISTORY OF FRANKIA RESEARCH. 28 2.7.0 TAXONOMY OF FRANKIA 30 2.8.0 TAXONOMY OF THE SPECIES 32 2.9.0 FRANKIA-THEMICROSYMBIONT 36 2.9.1 Morphology and anatomy 36 2.10.0 SYMBIOTICINTERACTION-APROCESS 37 2.11.0 ISOLATION AND STRUCTURE OF FRANKIA IN PURE CULTURE 39 2.11.1 The hyphae 40 2.11.2 The sporangia 40 2.11.3 The vesicle 41 2.12.0 GENETIC DIVERSITY OF FRANKIA 42 2.13.0 PROBLEM IDENTIFICATION 43 CHAPTER- III 3.0.0 MATERIALS AND METHODS 45 3.1.0 THE STUDY AREA 45 3.2.0 SURVEY OF STUDY AREA 46 3.3.0 ENVIRONMENTAL STUDIES 47 3.4.0 ROOT NODULE QUANTIFICATION 51 3.5.0 GENDER DIFFERENTIATION 51 3.6.0 GERMPLASM COLLECTION 51 3.7.0 SOIL ANALYSIS 52 3.8.0 AGROTECHNIQUES 53

3.8.1 Seed germination performance 53 3.8.2 Propagation through cuttings 54 3.8.3 Layering 54 II

3.9.0 ANTIOXIDANT ANALYSIS 55 3.9.1 Chemicals and reagents 55 3.9.2 Plant material extraction 55 3.9.3 Determination of plant extract yield 55 3.9.4 Determination of total phenolic content 56 3.9.5 Determination of total flavonoid content 56 3.9.6 Antioxidant properties of extracts in vitro 56 3.9.6.1 Free radical scavenging activity (DPPH method) 56 3.9.6.2 Reducing power assay 56 3.9.6.3 Scavenging of hydrogen peroxide 57 3.9.7 Statistical analysis 57 3.10.0 ISOLATION OF ENDOPHYTE 57 3.10.1 Washing 57 3.10.2 Surface sterilization 57 3.10.3 Isolation of Frankia in liquid culture 58 3.10.3.1 Technique I 58 3.10.3.2 Technique2 58 3.10.3.3 Technique3 59 3.10.4 Media for isolation 59 3.11.0 POST ISOLATION TASK 59 3.11.1 Incubation and sub-culture 59 3.11.2 Maintenance of culture 59 3.11.3 Decontamination 59 3.11.4 Nodulation test 60 3.11.5 Plant infectivity test 60 3.12.0 ISOLATION OF GENOMIC DNA 60 3.12.1 Isolation of genomic DNA from the root nodules 60 3.12.2 Frankia DNA purification 62 3.13.0 QUANTIFICATION 63 3.14.0 POLYMERASE CHAIN REACTION AMPLIFICATION 63 3.14.1 Primer 64 3.14.2 The primer stock 64 3.14.3 Annealing temperature 65 3.15.0 PCR REAGENTS 65 3.15.1 Taq DNA polymerase and assay buffer 66 3.15.2 Deoxyribonucleotide triphophate (dNTP) mix 66 3.16.0 DNA DILUTION 66 3.17.0 PCRMIX 66 3.18.0 PROGRAMME FOR PCR AMPLIFICATION 66 3.19.0 AGAROSE GEL ELECTROPHORESIS 67 3.20.0 GEL PHOTOGRAPHY 67 3.21.0 RESTRICTION FRAGMENT LENGTH POLYMORPHISM 67 3.21.1 PCR-RFLP data analysis 68 3.22.0 NUCLEOTIDE SEQUENCING 68 3.23.0 SEQUENCE BLAST 69 3.24.0 SEQUENCE SUBMISSION TO GENE BANK 69 3.25.0 PHYLOGENETIC ANALYSIS 70 iii CHAPTER-IV 4.0.0 RESULTS AND DISCUSSIONS 71 4.1.0 SOME SURVEY FACTS 71 4.1. I Land use and land cover 71 4.1.2 Elevation 75 4.1.3 Aspect 76 4.1.4 Slope 76 4.1.5 Vegetation analysis 78 4.1.6 Soil analysis 82 4.1. 7 The plant morphology 83 4.1.7.1 Leaf 85 4.1.7.2 Flower 86 4.1.7.3 Fruits and seeds 86 4.1.7.4 Root 87 4.1.8 Root nodule quantification 88 4.2.0 GROWTH OF HOST PLANT 91 4.2.1 Seed germination 91 4.2.2 Germination performance using active substances 93 4.2.3 Propagation by cuttings 94 4.2.4 Layering 96 4.3.0 ANTIOXIDANT 97 4.3.1 Plant yield 97 4.3.2 Total phenolic and flavonoids content 97 4.3.3 Antioxidant activities 98 4.3.4 Free radical scavenging activity (DPPH method) 98 4.3.5 Reducing power assay 100

4.3.6 H20 2 radical scavenging activity 100 4.3.7 Correlation study 100 4.4.0 ISOLATION OF FRANKIA 105 4.4.1 Surface sterilizat:on 105 4.4.2 Culture media for isolation 106 4.4.3 Nodulation test 106 4.4.4 Frankia under light microscope 106 4.4.5 Plant infectivity test 108 4.5.0 ISOLATION OF DNA OF FRANKIA FROM ROOT NODULES 108 4.5.1 Frankia DNA purification 109 4.5.2 DNA quantification 109 4.6.0 POLYMERASE CHAIN REACTION 111 4.7.0 PHYLOGENY AND GENETIC DIVERSITY 112 4.8.0 PCR-RFLP DATA ANALYSIS USING POPGENE SOFTWARE 113 CONCLUSION 126 REFERENCES 132 PUBUCATIONS 160 APPENDIX AI Tables

1.1 Species and sub-species of Hippophae L. 2 1.2 Overview of systematic treatment of Hippophae L 3 1.3 Composition of vitamins content of sea buckthorn etc. 5 1.4 Food, Medicine etc. from sea buckthorn 6 2.1 Distribution and habitat of representative genera of actinorhizal plants 25 2.2 Classification of Actinorhizal plants 26 3.1 Concentration of different chemical constituents used for PCR mix 65 3.2 Restriction enzymes showing their respective cutting sites 68 4.1 Vegetation classification using GIS ERDAS 72 4.2 Area covered in ha by different elevation revealed through GIS 74 4.3 Area covered in ha by different aspect revealed through GIS 75 4.4 Area covered in ha by different slope revealed through GIS 75 4.5 Field data showing different geo-physico-chemical parameters 79 4.6 Phyto-sociological characterization of vegetation in Lachen valley 80 4.7 Results of Soil Analysis. 81 4.8 Biophysical data of H. salicifolia related to nodule quantification 82 4.9a Biophysical parameters of H. salicifolia from riverine areas 85 4.9b Biophysical parameters from non-riverine areas 85 4.10 Germination performance of Hippophae salicifolia seeds 89

4.11 Effect of GA3 on root and shoot growth of sea buckthorn seedlings 90 4.12 Response of chemicals in germination of sea buckthorn seeds 90 4.13 Response of growth l)ormones in successful formation of callus 92 4.14 Plant yield v/s Plant Part. 98 4.15 Concentration of Phenols and Flavonoids in different extracts 98 4.16 Percentage inhibition (%1) of radical scavenging of male leaf 102 4.17 Percentage inhibition (%1) of radical scavenging of female leaf 102 4.18 Percentage inhibition (%!) of radical scavenging of male bark 102 4.19 Percentage inhibition (%1) of radical scavenging of female bark 102 4.20 Radical scavenging activities of BHT as standard 103 4.21 Quantification of DNA isolated from the root nodules 105 4.22 Diversity analysis of the partial 16S rRNA sequences GenBank accession 116 4.23 Sequences used in phylogenetic analysis 119 4.24 Genetic polymorphism of Frankia 120 4.25 Nei's measure of genetic distance (RFLP) 120 Figures

1.1 Natural habitat of Hippophae salicifolia 7 1.2 H. salicifolia plants 8 1.3 Fruits and seeds of H. salicifolia 9 1.4 Root nodules in different forms 10 1.5 H. salicifolia as sustainable means 11 1.6 Distribution of Hippophae L. in Europe and Asia. 12 3.1 Location map of study area in Indian sub-continent 47 3.2 Map (closer view) of study area 48 3.3 Sample datasheet used for field studies 49 3.4 Graphical representation PCR cycles 65 3.5 EMBL format used for submission of Frankia sequence 69 4.1 Ecological maps of the study area using 73 4.2 Pie chart showing %age of area covered by different land forms 74 4.3 Graph representing IVI sequence of vegetation 81 4.4 Quantification graph from riverine and non-riverine areas 88 4.5 Germination of seeds in different soil based media. 91 4.6 Different stages of growth raised from treated seeds 92 4.7 Treated cuttings raised in different soil compositions 95 4.8 Reducing power assay of leaves and barks 99 4.9 Correlation betwe.en DPPH and total phenol 101 4.10 Transverse section of root nodule under light microscope 110 4.11 Microscopic view of Frankia strains isolated from pure culture 111 4.12 Gel electrophoresis of PCR product of Frankia 113 4.13 Partial 16S rRNA sequence of uncultured Frankia 114 4.14 Phylogenetic dendrogram of Frankia sequence 115 4.15 PCR-RFLP of Frankia DNA 117 4.16 PCR-RFLP dendrogram 118 Chapter 1 Introduction

Sea buckthorn the general English term at Table 1.2. given to genus Hippophae was All the genera belonging to classified m 1753 m "Speciae Elaeagnaceae have nitrogen-fixing Plantarum" by Karl von Linne at the bacteria in their root nodules (Rousi, position 1023 (Proorocu, 2009). 1971; Soltis et al., 1995). The name The genus belongs to the family Hippophae (Latin words Hippo means Elaegnaceae, Order: Elaeagnales, horse and Phaos means to shine) Super order: Celastraneae, Subclass: reflecting its uses in ancient Greece Rosidae, Class: Magnoliopsida and (Bhattari, 2003). fu ancient Greece, the Division: Magnoliophyta (Rajchal, horses gained weight and attained 2009). It is a small family with five shiny coat after people used Hippophae species viz. Hippophae salicifolia D. as fodder (Subedi and Adhikari, 2001).

Don, H. rhamnoides Linn., H. tibetana The genus is locally termed as Tarobo Schlecht, H. neurocarpa S.W. Liu et (Bhutia), Ree-singri (Lepcha) and T.N. He, and Hippophae gyantsensis Amilchi/ Tarechuk/ Glzanguru/ Aclzuk Lian (Rousi, 1971). Among these, H. or Daile clzuk (Nepali) in Sikkim (fig. rhamnoides has further been sub­ 1.2 and 1.3). Siberian Pine apple, as divided into eight sub-species referred by the Russians, IS a (Rongsen, 1992; Schroeder and Yao, fascinating, eco-friendly, cold 1999; Bhattarai, 2003) (Table. 1.1). Himalayan plant. It is native to Europe Other genera of Elaeagnaceae are and Asia, has been known and used by Elaeagnus L. with 40 species and humans for centuries (Rongsen, 1992; Shepherdia L. with only 3 species Dhaka!, 2001). (Mabberley, 1998). Classification put Sea buckthorn (Hippoplzae L. ), 2n=24 forward by different authors from time (Li and Schroeder, 1996) is mainly to time for this genus may be referred INTRODUCTION 2

Table 1.1: Species and sub-species of Hippophae L. as per recent taxonomical studies. Genus: Hippophae Linn. Family: Elaeagnaceae. Species: Group Species Sub-species Coatless Hippophae salicifolia D. Don Hippophae rhamnoides Linn rhamnoides Rousi Sinensis Rousi carpatica Rousi caucasica Rousi turkestanica Rousi mongolica Rousi yunnanensis Rousi fluviatilis Rousi Coat Hippophae tibetana Schlecht Hippophae neurocarpa S.W. Liu et T.N. He Hippophae gyantsensis (Rousi) Lian used for economic and ecological (Rongsen, 1990). The whole plant purposes. It is a dioecious, nitrogen (fruits, roots, leaves and stem) is fixing, actinorhizal, wind-pollinated economically important (Fuhen et al., plant (Jeppsson et al., 1999; Arne 1999).

Rousi, 1971 ), deciduous, thorny willow About 14 million hectare or more of -like pioneering, shrubby or big tree up natural sea buckthorn orchards are to 10 m in height. The genus has been distributed widely over the world reported to grow in low humid (15%), (Rongsen, 1992) ranging between 27°- alluvial gravel, wet landslips, various 690 N latitude and 7° W to 122° E soil conditions, hills, gully tops and longitude (Rousi, 1971; Pan et al., riverside (flg. 1.1 and 1.2) with brown 1989; Yu, et al., 1989) spreading over rusty-scaly shoots (Rongsen, 1992; 3500 krn from east to west (Rongsen, Banjade, 1999; Basistha, et al., 2009a). 1990). Generally the global distribution Hippophae species are fast growing pattern of sea buckthorn shows that the (Rongsen, 1992), hardy woody plant plant is concentrated mostly in the cold often used in prairie conservation temperate regions of Hindu-khush programmes (Schroeder, 1988), which Himalayas (Rongsen, 1990; Subedi, is able to grow and survive well with 2007), parts of Europe and former low precipitations (300 mm), in soils USSR as well ·as Scandinavian region with pH of 9.5 and 1.1% salts (42 countries) (flg. 1.6). Sea buckthorn INTRODUCTION 3

Table 1.2: Overview of systematic treatment of Hippophae L. Taxa marked with an asterisk (*) were used in earlier classifications, but have never been validly pub­ lished or transferred. (Source: Bartish et al., 2002). Servettaz Rousi Avdeyev Lian & Hyvonen Lian et al., (1908) (1971) (1983) Chen (1993) (1996) (1998)

Hippophae Hippophae Hippophae Sect. Hippophae Sect. rhamnoides rhamnoides rhamnoides Hippophae rhamnoides Hippophae ssp.§ ssp. ssp. H. ssp. H. rhamnoides carpatica rhamnoides rhamnoides carpatica rhamnoides ssp. ssp. ssp. ssp. ssp. ssp. carpatica Salicifolia caucasica salicifolia carpatica caucasica ssp. ssp. ssp. ssp. ssp. caucasica Tibetana jluviatilis caucasica jluviatilis ssp. ssp. ssp. ssp.jluviatilis gyantsensis fluviatilis gyantsensis ssp. ssp. ssp. ssp. mongolica gyantsensis neurocarpa mongolica ssp. ssp. ssp. ssp. rhamnoides mongolica mongolica rhamnoides ssp. sinensis ssp. ssp. ssp. sinensis turkestanica rhamnoides ssp. H. salicifolia ssp. tibetana ssp. turkestanica turkestanica ssp. H. sinensis ssp. ssp. yunnanensis turkestanica yunnanensis ssp. H. salicifolia H. salicifolia sinensis* ssp. ssp. Sect. yunnanensis salicifolia Gyantsensis * Sect. ssp. sinensis H. Gyantsensis goniocarpa H. ssp. ssp. goniocarpa* yunnanensis goniocarpa* H. ssp. gyantsensis litangensis* H. H. neurocarpa gyantsensis H. tibetana H. neurocarpa ssp. neurocarpa ssp. stellatopilosa * H. tibetana §ssp=sub-species plant can tolerate temperatures of - different species of Hippophae (viz. H. 6o•c and do not wither at a heat of rhamnoides L., H. salicifolia D. Don 4o•c (Rongsen, 1992). In India, three and H. tibetana Schultz.) are naturally INTRODUCTION 4 distributed in high altitude areas of plantation m the world (Subedi and Himachal Pradesh, (Lahul Spiti, parts Adhikari, 2001). Presently, the largest

of Chamba, Kinnaur, Kullu, Shimla plantation IS located m and Kangra), Jammu & Kashmir (Leh Guskhrustalnyy, Vladimir State in the and Ladhak) and parts of Uttar Pradesh European part of former USSR (Singh et al., 1995; Singh, 1998) and (Rongsen, 1992). Being one of the Sikkim (Basistha and Adhikari, 2003). important species for forestry and also In Sikkim Himalayas, the plant grows in upgrading the sustenance of on the riverside, land slide areas, common people, sea buckthorn is being torrential slides and towards south-east used in the most different ways and is facing aspects in Lachen and Lachung considered as the plant of the 21" valleys of North Sikkim. H. salicifolia century.

D. Don, in Sikkim, grows generally at Sea buckthorn is propagated using altitudes ranging from 7844-10207ft. different means of agro-techniques like Sea Buckthorn prefers colonizing in seed propagation, cuttings, layering in open habitats (Singh et al., 1995). different soil conditions etc. (Auauzato Among all Hippophae species, H. and Megharini, 1986; Singh et al., salicifolia succeeds fast and can grow 1995; Basistha et al., 2009b ). The in various soil conditions with low seeds retain their viability after indoor nutrients (Subedi, 2007). storage for three to four years. Under

Estimates have shown that sea suitable conditions, they will germinate buckthorn plantation can fix 45 kg of during any season of the year (source: Baker Dwight, www.winrock.org/ N2 per hectare land, which is double forestry/factnet.htm). the amount of N2 fixed by Soya-bean plants (Rongsen, 1990). Sea buckthorn In central Asia, one-two years old sea­ has life span of 60-350 years (Singh, buckthorn plants raised from seeds 1998). were used in complementary and

Sea buckthorn being one of the comparative study of morphological, important crop, its propagation and physiological and biochemical features, agro-techniques had started way back after harvesting. In 1940s sea in 1960 by the Russian scientist who buckthorn was planted for soil collected and cultivated five varieties conservation, fuel, fodder and as wind of the genera and perhaps was the first break, apart from its medicinal and INTRODUCTION 5 commercial importance (Rongsen, Sea buckthorn is rich source of 1992). However, the level, quality and medicinal property, nutrients and quantity could be substantially met antioxidants. The fruits and seeds (fig. through scientific plantations and 1.3) are rich source of vitamins, managements. Demand for sea especially vitamin c (300-1600 buckthorn fruit and oil by the industries mg/100gm, which is 04-100 times has led to raise better quality of plants more than any vegetable fruit), with high economic value. Therefore, minerals, organic acid (2-4%, propagation using cuttings and other especial! y mallie acid which is higher vegetative means through selective than Citrus sp.1-2%), essential oils plantations fulfils economic and (unsaturated fatty acids around 86% ), ecological demands. Palmitic acid (about 34%), oleic acid

In the present study, different metho<;is (about 32%) and palmitoleic acid of propagation of Hippophae salicifolia (about 26% ), etc. and possess a number were studied in controlled laboratory of different medicinal properties like conditions, like germination enhancing micro-circulation of bloods performance of seeds in different soil capillaries and nourishes skin/hair, based media, response to different combats cardiac problems, etc. They concentration of growth regulators in also have great potential for seed germination, callus formation and preparation of health foods and a rooting in layering, softwood and variety of medicines (Centenaro et al., hardwood cuttings. 1977; Ansari, 2003; Zeb, 2004; Rongsen, 1992; Singh, 1998; Singh et Variations in habitat of different sea al., 2001) (table 1.4). It is considered to buckthorn species bring variation in the be a wonder plant known to be chemical components (Wang, 1990). economical! y viable for the rural

Table 1.3: Composition of Vitamins content of sea buckthorn and other fruits and vegetables (mg/lOOg).

Items Vit.A Vit.B1 Vit.B2 Vit.C Vit.K. Orange 0.55 0.08 0.03 50.0 Sea buckthorn 11.00 0.04 0.56 300-1600 100-200 Tomato 0.31 0.03 0.05 8.0 Kiwi fruit 100-470

Source: Occasional paper No. 20, 1992, JCJMOD, Kathmandu. INTRODUCTION 6

Table 1.4: Food, Medicine and Cosmetics, Beauty cream made from sea buck­ thorn in Russia, South Asia and Tibet Items Products Fruit juice Syrup juice, Carbonated juice, Sweets, wine, Champagne Bears, Jam, Tea, Dye, Chocolates, etc. Medicine Cancer, Blood Pressure, Gastric Ulcer, Burns, Chemical danger, Plastic surgery, Cough, ageing, Memory loss, etc. Beauty cream Makes skin fair, Clean and delicate. Possess therapeutic efficacy on skin, wrinkle, Skin sclerosis, etc. Cosmetics Improves micro-circulation of capillary vessel, nourishes skin, hair and cares 16 tropical diseases of scabies, etc.

Source: Occasional paper No. 20, 1992, JCJMOD, Kathmandu population in many ways (Singh et al., matured sticks/branches of these trees 1995). Table 1.3 shows the are used for fencing work and making composition of vitamin content of sea hedges around apple orchards and buckthorn and other fruits and vegetable gardens. The plants are also vegetables for reference. used as fodder and frrewood (fig. 1.5).

The leaves too are rich in nutrients, Sea buckthorn is also a good source of antioxidants and bioactive substances firewood (Rongsen, 1990). It IS (Rongsen, 2003; Small et al., 2000). apparent from the above discussions The young leaves contain high nutrient, that Hippophae fruits, leaves and barks carotenes and flavonoids. Generally, have potential in medicine, cosmetic vitamin C content in leaves is higher health and food products. However, than the fruit. It is also used as one of there has been no report on antioxidant the most important raw materials for and medicinal properties of Hippophae the extraction of vitamins and salicifolia growing in Lachen and flavonoids. Sea buckthorn leaves also whole of Sikkim. Therefore, the controls the growth of cancer cells in present study also aims to study the liver (Zhao et al., 1999). antioxidant property of fruit, leaves and barks of both male and female plants of In case of Sikkim, fruit juices are this species. traditionally used in dyes, making pickles, jam and treating some Sea buckthorn is used as a measure common ailments like cough and cold. species for soil conservation because of The fruit pulps are also used to treat its well-developed tap root system fever, diarrhea, scabies, constipation (Nepal et al., 2000; Rongsen, 2003). and other intestinal disorders. The The genus has extensive sub-terranean INTRODUCTION 7

Figure 1.1: Natural habitat of Hippophae salicifolia D. Don. A & B: No n-ri verine habitat and C & 0 : Ri verine habitat rooting system with trong soil binding actinomycetes known as Frankia in its ability, soil stabilization, riverbank root nodules (fig. 1.4 ). Hence, it can be control and water retention (Li, 1999) planted even m marginal soils and in reclamation of marginal land (Akkerman et al., 1983; Dobritsa and (Bantle er a/., 1996; Schroeder, 1988; Novik, 1992; Rongsen, 1992).

Li and Schroeder, 1999). Tap root up Though people observed "bump" like to 3 m and hori zontal roots up to 6-10 structure in the root nodules of m have been ob erved (Jar a, 1998). leguminou plants, nitrogen fixation With it luxuriant foliage and trong was unknown to all until Hellriegel and root ystem, it can retain the soil from Welfarth (1886) first announced in ero IOn. (Berlin) Germany that roots nodules of

Sea buckthorn al o grows in area with pea utilize atmo pheric N2 for their compre ed soil (with les aeration growth . This landmark di covery and less water), and has symbiotic opened a new era for under tanding relationship with nitrogen fixing science behind N2 utilization by plants INTRODUCTION 8

Figure 1.2: Hippophae salicifolia D. Do n plants. A & B: Trees of the study area. C & D: Shrubby tree , male and female plants, respectively INTRODUCTION 9

Figure 1.3: Fruii.S and seeds of H. salicifolia . A & B: Green frui ts. C & D: Ripe fruits and E: Seeds

and microbes, apart from solving other symbiotic) has now been well explored confusions and controversies on by scientists for better understanding of sources of nitrogen for plants (Sen, this complex process of reducing 1996). nitrogen to ammonia with the aid of

The complex mechanism of fixing of enzyme nitrogenase, and further atmospheric nitrogen by the bacteria in assimilation to amino acids and other legume root nodules and other bio-molecules into biotic processes by microorganisms (both free living and various plants. INTRODUCTION 10

Figurel.4: Root nodule of Hippophae salicifolia. A: Nodule in natural habitat. B: Clump of nod­ ules adhered to the roots. C: The individual nodule lobe cluster and 0 : Scattered nodular lobe

Almost all the nitrogen fixing (legumes plant symbiotic bacteria), microorganisms share two properties in Cyanobacteria (blue green algae), common. One of course is the ability to Frankia (actinomycetes which fix nitrogen and the other is that they associates with mostly woody all are prokaryotes except Eriophorum flowering plants) and Azospirillum sp. vaginatum, artie sedge which prefers (loosely associated and colonizes on using organic nitrogen for its growth the epidermis of host species like (Sen, 1996). maize, wheat and rice) (Vande Broeak

The nitrogen fixing organisms can be et al. , 1993; Giller, 2001). Among all grouped into four groups' -rhizobia the symbiotic nitrogen fixer, the gram INTRODUCTION 11

Figure 1.5: H. salicifolia as sustainable means for different components of temperate ecosystem

+ve actinomycetes of the genus forests (Alnus, Casuarina, Shepherdia, Frankia (Goodfellow and Cross, 1984) Comptonia, Coriaria), coa tal area are more significant because of their and river banks (Elaeagnus, association with large number of Hippophae, Casuarina), (Swensen, woody dicotyledonous angiosperm ' 1996), slopes and gullies (Hippophae, known as the actinorhizal (actina from Alnus) either lone stands or ntixed actinomycete, and rhiza the plant root) (A lnus, Hippophae). Except Datisca, plant (Tjepkema and Torrey 1979; which is herbaceou , other actinorhizal Baker and Schwintzer, 1990), which plants are perennial dicotyledonou , includes Hippophae salicifo/ia too. woody shrubs or tree .

Actinorhizal plant can thrive m Eastern Himalaya region of India has variou types of climates and high biological diversity of ecosy terns like Arctic Tundra (Dryas), angiosperm . About 70% of INTRODUCTION 12 angiosperms have been documented Actinorhizal plants have gained from this region. It is also a region of importance globally in the recent past high diversity of actinorhizal plants because of their potential m (Pradhan, 1993). reforestation of anthropically degraded

Actinorhizal plants are pioneering areas like mines, spoils, dam dykes, species in the nitrogen-poor and clear-cut forests or over-exploited disturbed soils. They have been widely agricultural and producing timber, used in afforestation programmes and windbreak, fuels, fodder, ornaments agro-forestry ecosystems (Ruskin, (Dawson, 1990; Diem and 1984; Seiler and Johnson, 1984 ), land Dommergues, 1990; Kucho et al., reclamation, biomass production and 2010). The amount of nitrogen fixed by forest restoration (Benson, 1982), most of these plants is almost the same timber, fuel, etc. to that of leguminous plants (40-350 Kg Nlha/year) (Torrey, 1978) and few

LEGENDS • H. rhamnoldc.:s L. sub sp. rhumnoides. • H. rham L. sub sp. nuwotflls v. soest. Y H. rhsm l. sub sp. carpatlcs Rousl. Q H. rham L. sub sp. cat.:casica RouSi. '\iA 4 H. rham l. sub sp. mongolica Rous; V • H. mam L. sub sp. yunnanensJ3 Rousl. 1 H. rllam L sub sp. gyantsenesi!: Rousl. • H. rham l. sub sp. slnen11is Rousl. ~ H. rham l. sub sp. tudc.estanica Rousi. • H. fibetana SChelechtand. 0 H. sa11Citofls D. Don. J- H. mt!frocarpa S. W. Llu et T. N. He.

Figure 1.6: Distribution of sea buckthorn (Hippophae L.) in Europe and Asia [Source: Arne Rousi (1971); Lu Rongsen (1990)]. INTRODUCTION 13 species can even release up to 70% of report demonstrated that Frankia fixed N2 to the ecosystem (Schwintzer, strains constituted a previously not 1984). described group of soil actinomycetes

Rhizobium strains and Frankia strains that are facultative symbiotic with have marked differences in gram higher plants. This association among staining (Diouf et al., 2003). There is a the plant root and the endophyte was marked difference in the nodules termed as 'actinorhiza' by analogy to produced by Frankia in actinorhizal mycorrhizal associations (Tjepkema plants and the Rhizobium strains in and Torrey, 1979). leguminous plants. This is because The term Frankia has been in use since there is extreme diversity of the host long time when it was first coined in plants and the symbionts (Silvester et 1886 by J. Brunchorst to honour his al., 1990). The infection site in the mentor A. B. Frank (Quispel, 1990). leguminous roots are usually within The groups of microorganisms that endodermis and inner cortical cells induce root nodules, capable of fixing covered by vascular tissues, whereas in atmospheric N2 in non-leguminous actinorhizal roots, nodules normally plants are broadly classified under the have a central stele that has infected genus Frankia. Frankia are tissue adjacent to or around it. filamentous gram-positive bacteria of

Actinorhizal root nodules endophyte the family Actinomycetales. This could establish their identity only in the ecological significance has made this year 1964, after its prokaryotic microorganism one of the important structure in Alnus glutinosa and Myrica subjects for the researchers (Baker and cerifera was revealed (Sen, 1996). Schwintzer, 1990). After several experiments for many Successful isolation of Frankia from years, report on first strain of Frankia, the root nodules have been made from Cpll (now known as HFPCcll) was almost all actinorhizal plants except reported in 1978 by Torrey and his co­ Ceuthostoma, Kentrotham.nus, workers from Comptonia peregrinn Chamaebatia, Dryas (Benson & (Callal!am, et al., 1978) and the ability Silvester, 1993) and Adolphia. (Huss­ of the organism to re-infect the host Danell, 1997). Till date strains from 25 plant and re-establish the symbiosis genera of actinorhizal plants have been was confirmed (Lalonde, 1978). This isolated all over out of which only nine INTRODUCTION 14 genera showed to give effective et al., 1980; Zhang and Benson, 1992). nodules when plants of their hosts were They are encapsulated with lipids, re-inoculated. This may be due to non­ spherical shaped measuring 2-6 f.Im in infectiveness of Frankia on their genus diameter and laterally or terminal! y host or nodulations were not examined. attached to hyphae by a short

As a consequence of many research encapsulated stalk (Sen, 1996). In works, Frankia is now relatively well natural condition, Frankia morphology defined. Three cell types are produced may be modified due to variation in by Frankia, which are useful in presence and absence sporangia, their studying prokaryotic behavior of the size, shape and presence of vesicles. organisms. New species have been This morphological change is proposed, physiology and growth influenced by the host plant during characteristics of some species have symbiotic process (Benson and been studied in details and step towards Silvester, 1993). molecular and genetic studies are going The rhizospheric microflora has on. Study of genetic diversity at the become one of the important economic molecular level, of the available factors for tropical agro-forestry. Their strains, whose infectiveness and use in the improvement of plant effectiveness are yet to be understood, productivity with decrease in the use of has made these organisms more chemicals in the form of insecticides interesting for the studies of host­ and pesticides has started showing symbiont relationship and their good results (Okon and Hader, 1987). interactions at different phases of N2 The richness of Frankia diversity in the fixation. natural forest is governed by the

Under in vitro conditions, Frankia are richness of the actinorhizal species characterized by septate and tightly along with ecological and geographical interwoven hyphae, sporangia and factors like slopes, aspect, vesicles. The sporangia are precipitation, etc.( Chen et al., 2008). multilocular at terminal or intercalary After extensive review of Frankia by regiOns. Vesicles, the site of Newcomb and Wood in 1987, there has nitrogenase activity are developed only been significant development m when the culture media is either devoid understanding the microorganism, of nitrogen or less nitrogen (Tjepkema, particularly its vesicle structure. The INTRODUCTION 15 anatomy of root nodules reveal various root nodules and cultures always may shapes of vesicle from elongated to not be required for these studies. pear shaped or globose (Torrey, 1985). Development of PCR techniques using Cell component of Frankia are delicate Frankia specific primers to 16S rRNA in nature, particularly the extra-cellular genes, 16S-23S rRNA intergenic covering of vesicle, which has caused regiOn, nijD-nifK intergenic region some difficulty in structure analysis genes, etc. have been mostly applied in through electron microscopy. But all cases of Frankia isolates. (Benson techniques like freeze fracture and et al., 1996; Clawson et al., 1998; freeze substitution has eased the Jamann et al., 1993; Jeong et al., 1999; procedure of electron microscopy still Murry et al., 1997; Nalin et al. 1995; with some problems in chemically N azaret et al., 1991 ; Normand et fixed specimens (Newcomb and Wood, al., 1996). It has become easy for the 1987). systematic, evolutionary, diversity and

Ecological studies and identification of ecological stndy of different Frankia Frankia in nodules or soil has been species by sequencing the PCR difficult due to lack of reliable and amplified genes of these convenient procedures, but microorganisms (Olsen et al., 1986; development of molecular studies like Woese, 1987). From different Polymerase Chain Reaction (PCR), publications and references, it has been sequencmg techniques and DNA revealed that molecular approaches in hybridization in the recent past has the study of Frankia isolates both from made the whole task easier and culture (in-vitro) or nodules have been convenient for studying Frankia useful m explaining phylogenetic population and phylogeny. relationship within the genus or species. (Giovannoni et al., 1990; Presently, Frankia analysis from soil Mirza et al., 1992; Nazaret et al., 1991; (Myrold et al., 1990), culture isolates Nick et al., 1992). This technique is at (Simonet et al., 1991; Bosco et al., par obvious for defined molecular 1992; Normand et al., 1992) and phylogeny groupings of Frankia nodules (Simonet et a[., 1990; Nazaret (Clawson and Benson 1999; Mirza et et al., 1991) can be done through al., 1994a; Rouvier et al., 1996). molecular approaches. Frankia can directly be isolated from uncultured Specific primers and probes have been INTRODUCTION 16

developed due to increase in Frankia were necessary.

sequence database for PCR and in situ Frankia research is an ongoing process hybridization. Now amplification of of many workers since long time. specific segment of Frankia is also There may be procedural hurdles but available (Mishra et al., 1991). Due to procedures like molecular techniques these sophisticated process and may answer many unsolved questions techniques, ecological influences on in Frankia research. Using proven introduced or indigenous Frankia techniques plays a vital role in solving populations are being studied by some issues. Study of genetic diversity different researchers all over the world. of Frankia in its natural population is From the previous work on Frankia one of the interesting study using (Reddell and Bowen, 1985; Sougoufara molecular tools. The present et al., 1992), it is revealed that host has experiments and investigations were a significant role to play compared to taken up with the following objectives: the microsymbiont during symbiosis •Environmental studies of native but later experiments through Hippophae of Lachen Region (field physiological data indicates that there based study). is a direct involvement of the host •Soil analysis for micro-nutrient plant, but lacks molecular evidences studies of Hippophae growing area. (Bajwa, 2004). However, some ecological, agrotechniques and •Isolation and Characterization of antioxidant studies on the host plant Frankia associates with roots of native too were taken up in this study during Hippophae. the study of genetic diversity of •Isolation of genomic DNA.

Frankia to correlate with its host and •Study of genetic diversity of Frankia the ecological niche. However, it was through PCR and RFLP. felt that some more studies in this line Chapter 2 Review of Literature

2. 1.0 HISTORY OF HIPPOPHAE L. leaves and young branches of sea (SEA BUCKTHORN) RESEARCH buckthorn resulted in rapid weight gain

Hippophae L. commonl y known as sea and a shi ny coat in horse (Rongsen, buckthorn has been in the use of 1990). mankind smce ages. Tibetan medical Sea buckthorn was ethnically used by

1 'Tantras' called Sibuyidian mentions the Mongolians from l3 h Century that the Tibetan medical classic rGyud­ onwards. In 'A Selection of Mongolian bzhi (Four books of pharmacopoeia), Medicine', a 120 chapter book written written by Yu Tuo Yuan Dan Kong Bu by Losan Quepie, during the Quing that was completed during the Chinese Dynasty (1 821-1850), mentioned 13 Tang Dynasty (618 to 907 A.D.), chapters were solei y devoted to sea contains 84 different set of prescription buckthorn and its clinical effects prepared from sea buckthorn (Rongsen, (Rongsen, 1990). , -,. nt.C 1011 1992). The original version of rGyud­ Siberict'ns started cultivating sea bzhi is thought to have been written in buckthorn (H. mongolica) in 1930s 1 Sanskri t around the 4 h Century A. D. , (Kalinina and Panteleyeva, 1987). later translated into Tibetan language Apart from developing new varieties of and submitted to the royal court the plants, Russians' also developed (Tsarong et al., 1981) . Hi story of sea various medicinal preparations and buckthorn, botanically, can also be health products for the astronauts of traced back to the age of Vedas. Its Sputni k satellite. Since then the genera utilities were also mentioned in ancient was soon introduced to other parts of Greek philosophy such as Dioscorides Russia (former Soviet Union), and and Theophrastus. In ancient Greece, neighboring countries (Traj kovski and sea buckthorn was used as a dietary Jeppsson, 1999). supplement for horses. Feeding on

24109J REVIEW OF LITERATURE 18

plant breeding, Sweden perhaps is the Mongolians mastered the economic world's most diverse gene bank for importance of this genus for centuries.

Hippophae, which consists almost all It is believed that the deserted war the wild, cultivars and selections from horses of the Greek army, in the 12 different breeding programme B.C., survived and became even available in the world (Bartish et al., stronger and shiny after wandering for 2000). long time in sea buckthorn forests. It is Chinese and Russian researchers also believed that sea buckthorn was engaged in sea buckthorn research used as a diet for race horses by the have made considerable contributions. Greeks.

The success story in sea buckthorn In another legend, sea buckthorn leaves research and development programme were preferred diet of a Pegasus, the from Tibet, Mongolia and Russia, etc. winged horse. encouraged many Asian countries like In some ancient kingdom, execution of Nepal, Bhutan, India and Pakistan to convicts by dropping them in a boiling start their own sea buckthorn barrel of sea buckthorn oil, gave development programmes. chance for the convicts to survive. In India, research on sea buckthorn is Cosmonauts of Mir spaceship of being carried out in states like former USSR used sea buckthorn Himachal Pradesh, Jammu and creams to protect themselves from Kashmir (Lahul, Spiti, and Ladhak) cosmic radiations and as a supplement and Sikkim (Singh 1998; Basistha and of oxygen. Adhikari, 2003). 2.2.0 ANTIOXIDANT 2.1.1 Legends associated with sea PROPERTIES AND CHEMISTRY buckthorn In the process of economic Genghis Khan, the Mongol conqueror, development, with the increase in in the12th century ordered his annies income, human society tends to care to eat sea buckthorn berries, to improve more about their health. Therefore, stamina and prevent altitude sickness. demand for healthy herbal organic The Tibetan doctors first understood foods developed from various plants the value of sea buckthorn and used it has also increased. Production of more in the 8th century. The Tibetans and the efficient and productive food items by REVIEW OF LITERATURE 19 the researchers are on demand. One 1996) and some essential fatty acids such plant with multiple qualities is with strong antioxidant activities (Yan Hippophae L. (sea buckthorn). and Lin, 2000). These compounds have

Hippophae L. has been used as food, the ability to inhibit cancer, beverages and medicine by the humans atherosclerosis (a common form of since decades, especially in Russia and arteriosclerosis m which fatty Tibet (Y aug and Kallio, 200 I) Sea substances form a deposit of plaque on buckthorn has attracted attention world the inner lining of arterial walls) over due to its nutritional and (Visonneau, 1997) built up immune medicinal values (Xu et al., 1994; system and decrease obesity (Letchamo Beveridge et al., 1999). The diversity and Lobatcheva, 1997; Houseknecht, m biochemical and nutritional 1998) treating pulmonary, hepatic, expressions shown by this plant are due gastrointestinal and articulate diseases to its distribution, origin, climate and (Olziykhutag, 1969). Oils extracted methods of extraction (Wang, 1990; from the seeds can be successful! y used Zeb and Malook, 2009). for treating various kinds of irradiation, mucosa, ulcers and burns. It can also Antioxidants can react with free be used against stomach cancer at its radicals during the oxidation process initial stage (Yang et al., 1999; Yang by acting as a reactive species and Kallio, 2003; Rongsen, 1992). It is scavenger and liberating catalysts, so estimated that more than I 00 kinds of antioxidants can be used to reduce the bioactive and antioxidant elements oxidative process (Gulcin et al., 2005). have been identified in leaves, fruits, Bioactive compounds like ascorbic bark and seeds (Rongsen, 1992; acid, carotenoids, tocochromanols and Rongsen, 1993; Singh and Awasthi, phenols are antioxidants. Sea 1995; Singh, 1998; Singh et al., 2001) buckthorn, apart from being a and thus, has high values m repository of vitamins, minerals and development of medicine, herbal and essential bioactive substances (Xurong health care products (Lu and Ma, et al., 200 I), also has potential 2001). The polyphenols m sea antioxidant activity (Velioglu et al., buckthorn fruit have antioxidant 1998; Halvorsen et al., 2002; Nilsson properties and thus can be used for et al., 2005) attributed to its flavonoids , repairing damages caused by free oils, vitamin C (Li and Schroeder, REVIEW OF LITERATURE 20

radicals (Y ao and Tigerstedt, 1992; kaempferol, fatty acids, triacylglycerol, Yang and Kallio, 2001) phytosterols, sugar, organic acids,

The leaves possess anti-inflammatory proanthocyanidins and phenolic properties (Ganju et al., 2005; Padwad compounds (Abid et al., 2007; Fan et et al., 2006). Leaf extract is also used al., 2007; Li et al., 2007). Fruits in ointments for treating burns, skin contain globulins and albumin proteins, cracks, scabies, impetigo, keratosis and carotene, saturated and unsaturated cures xeroderma (Rongsen, 1992). The fatty acids, free amino acids, young leaves contain high nutrient, flavonoids and vitamin E (Rongsen, carotenes and flavonoids. Generally, 1993; Singh, 1998). vitamin C in leaves is higher than the Many works on nutritional attributes of fruit. It is also used as one of the most sea buckthorn has been carried out but important raw materials for the review of literature did not provide any extraction of vitamins and flavonoids. substantiation on antioxidant and Sea buckthorn leaves also controls the nutritional properties of Hippophae growth of cancer cells in liver (Zhao et salicifolia D. Don. There are few al., 1999). reports on antioxidants of leaves

Fruits of sea buckthorn are rich source (Geetha et al., 2003), seeds (Negi et of vitamin C (300-1600 mg/1 OOgm), al., 2005) and fruits (Eccleston et al., which is 04-100 times more than any 2002) of H. rhamnoides. Research on vegetable fruit, high amount of organic detailed chemistry and antioxidant acid (2-4% ), especially mallie acid property of H. salicifolia is perhaps, which is higher than Citrus sp. (1-2%) yet to be done. (Rongsen, 1992). Fatty acids, orgaruc 2.3.0 AGROTECHNIQUES OF acid, tannic acid (Singh, 1998), SEABUCKTHORN

Palmitic acid (about 34% ), oleic acid Hippophae L. (sea buckthorn), due to (about 32%) and palmitoleic acid its beauty, was introduced as an (about 26%) are the main constituents ornamental plant in botanical gardens of pulp oil. Higher quantities of of Europe. Though sea buckthorn was unsaturated fatty acids (around 86%) well known to the human society since are found in seed oils. Seeds also ages, plantation works through contain vitamin C, large amount of different means started only few carotenoids, vitamin E, flavonoids, decades ago. Its hydrophilous character REVIEW OF LITERATURE 21

(Ansari, 2003) helps the plant to grow 24°C-26°C (Ansari, 2003) with at places with 400-600 mm annual viability up to 3 years (Rajchal, 2009). rainfall (Rongsen, 1992; Ansari, 2003; Various techniques like scarification, Dhakal, 2001). Besides its economic hot water and rooting hormone and ecological qualities, sea buckthorn treatments have been employed for is also regarded as the "slave of propagation of sea buckthorn seeds but degraded land" (Constandache and research on use of growth regulators on Dinca, 2009). germination response could not be Realizing the importance of sea reviewed in literature for seed buckthorn in different aspect of human propagation. Though seeds may have life, including commercial interests, higher emergence rate (Rajchal, 2009), different agrotechniques and now a days, seed propagation is propagation techniques on this wonder basically used for breeding and plant has been carried throughout the introduction works, forest land world in recent years. H. salicifolia of reclamation, protective and ornamental Sikkim Himalayas is untouched of purposes and sometimes for root stocks major researches till date apart from its (Rongsen, 1992). illustration in the seven volumes of Despite of significant biological "Flora of British India" by Sir J. D. variations in sea buckthorn seeds from Hooker (1872-1897). Agrotechniques varying ecological and geographical of sea buckthorn through different areas, each population have individuals methods like cuttings, seed propagation with extended period of seeds (Auauzato and Megharini, 1986), germination (Bobodjanov and layering, etc. have been carried out in Kabulova, 2005). Many economic the past, but the response of plants in yields may be lost in the plant fruits different environmental conditions grown from seeds due to higher level have been found to be different. of heterozygosis. Therefore, cultivation Naturally, propagation of sea of sea buckthorn for economic uses buckthorn through seeds is the main needs propagation techniques where way for its occupation into new habitat genetic uniformity of the seedlings is (Lisenkov et al., 1969). This genus is ensured, keeping its integrity and also thermophilic, so the seeds can similarity with the selected mother germinate at a temperature between plant. REVIEW OF LITERATURE 22

H. rhamnoides L. is propagated both high yield (Li, 2002). Use of Nitrogen sexually and by vegetative means. fertilizer to the plants inoculated with Through vegetative propagation, plants Frankia, not only delays nodulation but can be propagated on hill slopes and also has adverse effect in nodulation rivers banks (Rongsen, 1990) with (Akkermans et al., 1983; Montpetit and proper spacing (1m within the row and Lalonde, 1988; Bosco et al., 1992). 4m between rows) depending on This was not the same with other terrain and male: female ratio between actinorhizal plants. When studied in 1:6 and 1:8), adequate manure and culture solutions, nodulation reduced at water supply (Li and Schroeder, 1999). lower pH in some actinorhizal plants Propagation establishment of H. including Alnus glutinosa. High rhamnoides, through hard and soft nodulation rate has been observed in wood cuttings, seed propagation (with soil pH between 5.5 and 7.2 In soil pH or without treatment of different of 4.5, the viability of endophyte also growth regulators) (Auauzato and decreased (Griffiths and. McCormick, Megharini, 1986; Ansari, 2003; 1984).

Basistha and Adhikari, 2003; Basistha H. salicifolia, despite of having large et al., 2009b; Rongsen, 1992; Singh population m Sikkim Himalayas, 1998) is well suited in well drained, systematic and scientific studies are deep, sandy loam soil with sufficient still immature for exploiting its true organic matters (Li and Schroeder potential. It remains underutilized due 1999; Li and Oliver, 2001 ).Propagation to lack of further research and through tissue culture (Lin et. al., development. 2007; Vescan et al., 2009) and layering 2.4.0 DISTRIBUTION OF (B asistha et al., 2009b) has also been ACTINORHIZAL PLANTS adopted in this genus. It was well established that genus Sea buckthorn can grow well in pH 6- Frankia was an actinomycetes and the 7. For general growth and fruit plants bearing such associations with production in future, soil acidity and Frankia were called "non-leguminous alkalinity (except at extreme levels), plants" until the term 'actinorhizal' may not be a limiting factor, but was coined in the First International requires adequate soil nutrients with Conference held at Harvard Forests in phosphorus for better fruit qualities and Petersham, Massachusetts, (USA) in REVIEW OF LITERATURE 23 April 1978, replacing the term "non nitrogen rich organic matter mainly in leguminous" (Tjepkema and Torrey, the forest soil and play primary role in 1979; Newcomb and Wood, 1987), and the dynamics and biodiversity of published in the proceeding titled terrestrial ecosystems.

Botanical Gazette: 140 (suppl.) in 1979 According to Baker and Schwintzer (Huss-Danell, 1997). Researches have (1990), 288 species with 24 genera also led to the understanding that belonging to 8 families of actinorhizal symbiotic relationship between the plants have been reported so far. filamentous gram +ve bacteria of the Similarly, Benson and Silvester (1993) order actinomycetales-Frankia and have reported actinorhizal plants fine roots of certain angiosperms form having 194 species with 24 genera, but the actinorhizal associations and the without details. However some reviews host plants involved in these kind of state that there are more than 200 associations are known as Actinorhizal species of actinorhizal plants, which plants (Tjepkema and Torrey, 1979; includes 25 angiosperm genera Huss-Danell, 1997). belonging to 8 families distributed Franlda, unlike Rhizobium, are among 4 orders viz. Fagales, filamentous, branched, gram +ve Cucurbitales, Fabales and Rosales actinomycetes, which have symbiotic (Soltis et al., 1995; Wall, 2000; association with large number of Schwencke and Caru, 2001). Of the 25 woody dicotyledons, whereas genera of actinorhizal plants reflected 1 Rhizobium is a unicellular gram -ve in some literatures, the 25 h genera bacteria that has symbiotic association might have been referred to Rubus, with only legume family plants which as per Bond (1976) and Becking excepting Parasponia of Ulmaceae (1984) is an actinorhizal genus. But family (Obertello et al., 2003). Stowers (1985) did not agree Rubus to

Actinorhizal plants form a key be a truly actinorhizal plant. Even component in natural ecosystem, agro­ Jeong and Myrold (2003) have ecosystem and agro forestry by reported that actinorhizal plants consist substantiating fixed nitrogen (1-150 kg of 24 genera, which is agreed by the N/haly) to these systems (Torrey, 1978; majority of researchers in this field Dawson, 1983; Russo, 2005). (Table 2.1 ). Actinorhizal plants are the source of Actinorhizal plants are woody dicots REVIEW OF LITERATURE 24 except Datisca sp., which is actinorhizal (Benson et al., 2004). herbaceous plant (Franche et al., 1998) Different authors have tried to classify and are found throughout the world actinorhizal plants with some accept Antarctica (Table 2.1). Alnus differences. The classification put and Elaeagnus species are found in the forward by Cronquist (1988) and hilly region of the tropics (Myrold, reported by Benson et al. (2004 ), 1994). These plants are pioneering reveals some interesting features species of the temperate regions (Table. 2.2). In Cronquist's (excepting Casuarina and Myrica), classification, actinorhizal plants are which prefer to grow in open areas placed under Magnoliopsida, where having sun facing aspects during their Rosales are not ascribed with the sub early succession. Due to their ability to class Harnarnelidales. Elaegnaceae and utilise atmospheric N2 through Rharnnaceae have been placed in Frankia, they show positive growth in different order other than Ro~ales, sandy and swampy soils with marginal Datiscaceae was assigned to Violales nitrogen level and range of (Dilleniidae) and Hamarnelidales, environmental stress (Dawson, 1990; Casuarinales, Fagales and Myricales Tredici, 1995) (may also refer to Table were combined together. In contrast to 2.1). the above, Benson and his co-workers 2.5.0 TAXONOMY AND presented classification of actinorhizal EVOLUTION OF ACTINORHIZAL plants in a modified way. Here, PLANTS Elaegnaceae and Rhamnaceae have

It is understood by now that been placed in the order Rosales. actinorhizal plants cover a wide range Similarly Datiscaceae has been of dicotyledonous plants, with fewer ascribed with order Cucurbitales m lineages in their taxonomic relations. contrast to Violales (Dilleniidae). In They have both ancient and advanced both the classifications, the order characters (Bousquet and Lalonde, Rosales are not ascribed with sub-class 1990; Sen, 1996). Actinorhizal plants Harnamelidales but, other sub-class are included in different families of placements are similar. which, all the genera of a particular Traditional classification of family may not be actinorhizal, except actinorhizal plants were done on the in Elaegnaceae where all plants are basis of morphological characters, REVIEW OF LITERATURE 25 Table 2.1: Distribution (after Baker and Mullin, 1992)* and habitat of representa- tive genera of actinorhizal plants Family Genus Native from following Habitat re ions Betulaceae Alnus North America, South bogs, riparian America, Europe, Northern Asia, Southern Asia

Casuarina- Allocasua- Australia* sand dunes, saline, ceae rina desert, coastal areas Casuarina Australia Gymnostoma Australia

Coriariaceae Coriaria Australia, North America, gravel, poor soils South America, Europe

Datiscaceae Datisca North America, Southern gravel streams Asia Elaeagnaceae Elaeagnus Northern Asia, North Poor and stressed America, Europe, Southern soils, disturbed sites, Asia hilly slopes Hippophae Europe, Northern Asia, River banks, torrential Indian sub-continent, Ti- and land slide areas, bet. fragile slopes, gullies, disturbed soil. Shepherdia North America

Myricaceae Myrica Southern Africa, North bog, ocean dunes America, South America, Australia, Southern Asia northern Asia Comptonia North America chaparral, upland

Rharnnaceae Adorphiab North America Ceanothus North America Colletia South America semiarid soils, sand Rosaceae Cercocarpus North America gravelly soil

Dryas North America the same Purshia North America Australia* Australia and/or Oceania •cruz-Cisneros and Valdes (1990). Source: Dommergues, Y. R, 1997 and Berry, M.A., 1994 with some personal modifications. which are distantly related and morphological classification, Mullin classified into four of the six major and An (1990) suggested that subclasses of angiosperms (Cronquist, symbiotic association must have 1981) (Table 2.2). As per this evolved several times during the course REVIEW OF LITERATURE 26

Table 2.2: Classification of Actinorhizal plants ".

Subclass b Order • Family Nodulation Genus ratio Hamamelidae Fagales Betulaceae 1/6 Alnus Casuarinaceae 414 Allocasuarina Casuarina Ceuthostoma Myricaceae 2/3 Gymnostoma Comptonia Myrica Rosidae Rosales Elaeagnaceae 3/3 Elaeagnus Hippophae Shepherdia Rhamnaceae• 7/55 Ceanothus Colletia Discaria Kentrothamnus Rosaceae 5/100 Retanilla Trevoac Cercocarpus Chamaebatia Dryas Purshiag Magnoliidae Cucurbitales Coriariaceae 1/1 Coriaria Dilleniidae Datiscaceae 1/1 Datisca "Compiled after Baker and Schwintzer (1990), Swensen (1996), Benson and Clawson (2000), and Schwencke and Caru (2001). b According to the classification of Cronquist (1988). 'According to the classification of the Angiosperm Phylogeny Group (1998); all of these orders fall in the 'Eurosid I' group of eudicots. • Number of nodulated genera over the total number of described genera in the family 'Adolphia may be actinorhizal, but has not been confirmed (Cruz-Cisneros and Valdes, 1991). 'Talguenea should be combined under Trevoa (Tortosa, 1992). "Purshia and Cowania have been combined under Purshia (Henrickson, 1986). of angiosperm evolution. Taxonomy of reports on pollen records, members of actinorhizal plants, though still unclear, Rosales, Protales and Rhamnales was their respective symbiotic association relatively advance but Fagales and could be homologous as they fall under Myricales might have evolved earlier the same subclass (Swensen, 1996). than other actinorhizal members (Sen,

Actinorhizal plants have originated in 1996). It is also theoretically estimated the late cretaceous period and have that present actinorhizal plants, before diversified to different ecological niche their divergence into families and their (Magallon et al., 1999). From the respective genera, might have come in contact with Frankia in a selective REVIEW OF LITERATURE 27 ecological niche that might have including Parasponia sp. (rhizobially favoured the evolution of symbiotic nodulated), together in a single clade association. called the "core rosid" (Chase et

Another theory of evolution presumes a/.,1993; Mullin and Dobritsa, 1996; that in the early cretaceous period, due Dawson, 2008). Soltis et al. (1995) to scarcity of available nitrogen (Bond, reported that of the four sub-clades that 1983) in the atmosphere, some woody, contain Nz fixing symbiosis, wind pollinated dicotyledonous plants actinorhizal plants were found in three. had to forcefully associate with They also suggested that all N2 fixing Frankia. But after this association, plants have originated simultaneously during the course of evolution, there along with several other non N2 fixing might have been increase in the plants. Restriction in nodulation atmospheric nitrogen and some activity in core rosids emerged with a changes on certain advantageous and hypothesis that the predisposition to favourable characteristics for symbiotic form nitrogen fixing root nodule association. So, some plants lost the symbioses emerged once during symbiotic ability with Frankia but their ang:tosperm evolution (Soltis et al., genetic makeup of their symbiotic 1995). association of the past existed. Today Now, it is also concluded that certain phylogenetic studies (Normand actinorhizal plants occur in 10 families and Bousquet, 1989) and DNA and are distributed among 5 of the 8 hybridization studies carried out on main lineages of the 'core rosid' group host plant (Bousquet et al., 1989) (Magallon et al., 1999). Swensen supports this theory. (1996) combined rbcL sequence

It is understood in the recent times that analysis with morphological and actinorhizal plants have been found anatomical characters and showed that more closely related through molecular this rosid clade of actinorhizal phylogenetic studies on large symbiosis originated at least four times (Bousquet and Lalonde, 1990; Sprent, samplings of symbiotic N2 fixing species. Molecular analyses of 1994), which is separated from other flowering plants based on rbcL gene lineages by related non actinorhizal have revealed distinctiveness and have plants. This combined characteristics placed both actinorhizal and rhizobia, of molecular (rbcL sequence), REVIEW OF LITERATURE anatomical and morphological data, glutinosa, and concluded it to be a after further investigation by different parasites growing in the roots of plants. workers, led to the conclusion that N2 In 1866, Wornin conducted anatomical fixing actinorhizal symbiosis should studies of root nodules where he have originated at least 3-6 times, observed hyphae passing through the independently (Roy and Bousquet, intracellular walls. The round vesicular 1996; Swensen, 1996; Swensen and swellings at the tips of these passing Mullin, 1997; J eonget al., 1999). hyphae were considered to be a fungal

From these facts, diversity of spore by him and named the organism actinorhizal plants have expressed as Schinzia alni due to its resemblance symbiotic predisposition in number of to parasitic fungus Schinzia cellulicolia times during their evolution (Clawson (Sen, 1996). et al., 2004). This is an indication that Professor A. B Frank, a Swiss these plants (Frankia and Rhizobium microbiologist, considered the root associated) had inherent capacity to nodules of both leguminous and non­ adhere symbiotically with soil leguminous plants as protein bodies microbes (Swensen and Mullin, 1997; and rejected the idea of presence of Jeong et al., 1999). microorganisms in it. The name

Classification approaches usmg Frankia subtilis was first proposed in molecular techniques for phylogenetic 1886 by J. Brunchorst to honor his studies have given new perspectives to mentor A. B Frank, after studying the actinorhizal plants, providing important cytological differences of non­ evolutionary and ecological insights leguminous and leguminous roots. (Benson and Dawson, 2007). Both Brunchorst and Frank considered the microorganism Frankia to be a 2.6.0 HISTORY OF FRANK/A fungus. After further studies, Frankia RESEARCH: was classified as actinomycetes by The study of root nodules of non­ Krebber in 1932 (Quispel, 1990). leguminous plants was shrouded in the Two publications to their discovery by mist of misconceptions for a long Hellriegel and Wilfarth during 1886- period. Meyen (1829) first described 1888 proved the legume root nodules non-leguminous plant nodule after could fix atmospheric nitrogen with the discovering it from the roots of Alnus help of bacteria residing in the cortical REVIEW OF LITERATURE 29 cell and proposed difference between result concluded that the two nitrogen users and nitrogen microsymbionts are two different accumulators, where Alders were microorganisms (Brewin, 2002). This included as nitrogen accumulators, observations along with cytological initially, brought an end to a !0-year­ differences among the leguminous and old controversy about sources of non-leguminous microsymbiont, led to nitrogen for growth of plants and understand that these two opened up new frontiers of plant microorganisms were different from microbial science (Bottomley, 1912; each other (Pawlowski and Bisseling, Quispel, 1988; Sen, 1996). 1996).

After a decade, experiment of The identity of the non-leguminous Hellriegel and Wilfarth was further microsymbiont was not established strengthened by Hiltner in 1896 by unless Krebber classified it as reporting the presence of some kind of actinomycetes in 1932 (Quispel, 1990) Bacteria in Elaeagnus and Alnus sp., and reported by Becking et al. (1964), who also proved and demonstrated that after viewing under electron young Alders without root nodules microscope. It was only after this; the cannot survive in N2-free soil but when presence of actinomycetes in the root inoculated with root nodule nodule of non-leguminous plant was supplements, gave positive results. In established. the year 1907, Hiltner also reported Further detailed stndy could be carried nitrogen fixing organisms in the root out only after isolation of this nodules of Cycas (Bottomley, 1912). microsymbiont in pure culture by These experiments led to comparison Torrey and his co-workers (Callaham of leguminous and actinorhizal root et al., 1978). After successful pure microsymbionts and concluded that culture isolation of Frankia was carried actinorhiza were not fungus but out, a classical reviews by Newcomb bacteria. and Wood (1987) and Benson and In another study, Beyerinck (following Silvester (1993), illustrated vivid the steps of Koch and Pasteur) isolated descriptions of structure and ultra­ bacteria from legume root nodule but structure of Frankia cells through when he again tried to infect the root of electron microscopy. But, Benson and non-leguminous plant, it failed. This Silvester (1993) have also discussed REVIEW OF LITERATURE 30 some difficulties in studying Frankia On the basis of host specificity, most of with electron microscope because of the Frankia were divided into four .the delicate nature of some cellular groups: (1) Alnus and Myrica, (2) components. Casuarina and Myrica, (3) Myrica and

After this important historical research Elaeagnus, and (4) members of on Frankia, further researches Elaegnaceae (Elaeagnus, Hippophae gradually took place and are still going and Shepherdia) (Baker, 1987). This on. classification was considered to be incomplete because it was not possible 2.7.0 TAXONOMY OF FRANK/A to isolate Frankia from all actinorhizal Classification of Frankia was initiated plants and some strains in pure culture even earlier than pronouncing the term showed non-infective character and 'actinorhiza' in place of the term 'non­ could not re-nodulate their hosts. legume', for nodule bearing plants, in Further some strains also lacked the first conference on Frankia and morphological, cytochemical and actinorhizal plants (Tjepkema and physiological criteria assigned to Torrey, 1979), mentioned earlier. Frankia (Hahn et at., 1989; Before successful isolation of the Lechevalier, 1994). However, based on endophyte, Becking (1970), after phylogeny (Lechevalier and redefining the genus, placed it as a Lechevalier, 1989), DNA homology member of the order Actinomycetales (An et at., 1985) and serology (Baker in a new family, Frankiaceae. Using et at., 1981), Frankia were divided in crushed nodules as inocula and host two sub-groups -A and B. specificity (Lechevalier, 1994), he Only after isolation of different strains further went ahead and put up a of Frankia in pure culture, a new proposal of establishing 10 species of dimension on Frankia taxonomy this genus (Frankia alni, F. elaeagni, started emerging, especially when it F. brunchorstii, F. discariae, F. was found that individual Frankia casuarinae, F. ceanothi, F. coriariae, strains could nodulate plants of F. dryadis, F. purshiae, and F. different orders and were not so rigid cercocarpi). Since isolation of m host specificity (Benson and organism had failed number of times, Silvester, 1993), analogous to he named the members as "obligate Rhizobium. Many Frankia strains have symbiotic organisms." REVIEW OF LITERATURE 31 been isolated and attempts have also strains reacted with the rRNA of been made to classify them. Nacordioides albus. However, this

It may be noted that only one genus could not be considered. and no species of Frankia had been In another experiment, when Hahn et recognized till 1988, this may probably al. ( 1990) tried to exploit sequence be because of differences m position 180 to 240 as a probe, it morphological and physiological reacted with all the strains tested but at characters of the genus. In Bergey's higher temperatures some strains failed Manual of Systematic Bacteriology, to hybridize, which brought difference val. 4 (1989), Frankia are included in the sequence of other strains. Out of among "actinomycetes with 22 strains only two (Actinomadura and multilocular sporangia" (Lechevalier Microbispora) tested reactive. This too and Lechevalier, 1989). Frankia cannot indicated lack of probe specificity. be classified on the basis In a new approach to characterize morphological characters because they genus specific Frankia, Simonet and are too diverse to identify (Weber et his co-workers in 1991, designed two a/., 1988). sets of primers and tried to solve Classification attempt at molecular through Polymerase Chain Reaction level was initiated. It was widely (PCR). The first one was universal believed that hypervariable regions of primer targeted to nitrogen fixing 16S rRNA may help in phylogenetic bacteria, which gave negative result for classification at genus or species level non-nitrogen fixing bacteria. The (Harry et al., 1991). Hypervariable second set of primer was targeted to region of 16S rRNA genes of 1020 to specific nifH-nijD region of Frankia. 1042 (E. coli numbering) for Frankia Using these primers when DNA was taxonomy differed in comparison with amplified, genus specific length rRNAs of effective (N2 fixing) and fragments was achieved. These infective Frankia strains (Hahn et al., fragments thus generated were Frankia 1989). Probes containing 20-22 specific and also related to nucleotides targeting this region failed Geodermatophilus (closely allied to interact with other actinomycetes put genus with Frankia). To separate the in experiment but both types of probes two genera, again two primers were from effective and infective Frankia designed to amplify 16S and 23S REVIEW OF LITERATURE 33 1990), host specificity (Bosco et at., genomic spec1es as Elaeagnaceae 1992), phylogenetic study of compatible and one genomic species as carbohydrate uptake (Ganesh et at., Casuarina compatible. Fernandez et at. 1994), etc. with mixed success. In mid (1991), further reported on the study of

80s and 90s of last century, molecular hypervariable region E2 of different approaches like DNA-DNA relatedness isolates of Alnus, members of analysis, (An et at., 1985), Low­ Elaeagnaceae and Casuarinaceae Frequency Restriction Fragment conducted by Normand and his co­ Analysis (LFRFA) (Beyazova and workers, where he had confirmed Lechevalier, 1992), 16S rDNA and 16S classification of isolates from Alnus, rRNA sequences (Nazaret et at., 1991; into genomic species through DNA­ Normand et al., 1996; Clawson et at., DNA relatedness, but varied with 2004), arbitrary primers (Sellstedt et ribosomal sequence of strains of at., 1992), nitrogen fixation (nif) genes Elaegnaceae genomic species. Later (Jeong et at., 1999) and glutamine Nazaret et al. (1991) compared all 9 synthetase (Clawson et at., 2004 ), etc. genomic sequences using PCR have been approached with some technique to amplify 268 bp DNA interesting facts in the molecular level. segment of 16S rRNA gene, where

DNA-DNA hybridization study for except one; all the strains from one Frankia classification taken up by An genomic species had same sequences et at. (1985) was perhaps the first of its and were also different to other kind and found high relatedness among genomic species. most of the Frankia strains isolated Similar studies on DNA-DNA from Alnus sp., but could not relate hybridization or nucleic acid isolates of other species like hybridization, using 16S rRNA-23S Elaeagnus, Casuarina, etc. Similar rRNA as molecular markers (Simonet study on diversity among 43 isolates of et at., 1994; Benson et at., 1996; the genus Frankia was also conducted Normand et al., 1996; Clawson et al., by Fernandez et at. (1989). In his 1999; Ritchie and Myrold, 1999), use study, out of these isolates, at least nine of PCR amplified 16S rRNA partial compatible genomic species among the sequences from pure culture or strains were classified as three genomic uncultured endophytes from the root species as Alnus compatible, five nodules (Mirza et al., 1994; Simonet et REVIEW OF LITERATURE 34 al., 1991; Simonet et a/.,1994) or researchers took up the work with probing PCR products with specific different procedures to attain forward and reverse primers to identify homologous results. specific Frankia populations, in dot Various rr10lecular procedures like blots (Mirza et al., 1994b) and in PCR RFLP analysis (Akimov and Dobritsa, assay (Simonet et al., 1994,) have been 1992) of PCR amplified 16 S rDNA carried out in the past. Nazaret et al. (Huguet et al., 2001), IGS of 16S-23S (1991), also constructed a phylogenetic rRNA operon (Rouvier et al., 1996; tree from the sequences described by Ritchie and Myrold, 1999), IGS of nifH Fernandez et al. (1989), to measure -D (nitrogenase) genes (Cournoyer and phylogenetic relations using 16S rDNA Normand, 1994) nifD-K genes (Jaman sequences, which revealed Alnus et al., 1993; Nalin et al., 1997) and infective, Elaeagnus infective and glnll gene (Cournoyer and Normand, Casuarina infective groups Ill 1994) have been applied to classify respective clusters. Frankia from pure culture or nodules. The analysis of DNA using PCR Apart from these procedures, Beyazova techniques allowed the scientists to and Lechevalier ( 1992) used pulse field study the microbes with or without electrophoresis to separate bigger culturing it. This proved to be one of molecular weight fragments spliced the powerful tools in the study of with restriction enzymes having less ecology and classification of Frankia. number of restriction sites and named it Along with applications of PCR Low Frequency Restriction Fragment technique used in the study of Frankia, Analysis (LFRFA) and confirmed that Restriction Fragment Length F. alni sub-sp. pommeru clusters Polymorphism (RFLP) of nif complex differently than other Frankia strains (nifA-B, nifK and nifH) has added a isolated from Alnus sp. new dimension in classification and Use of arbitrary primers during PCR phylogenetic study of the genus. (Sellstedt et al., 1992) and certain Initially, Nazaret and his research team primers meant for consensus motifs of m 1989 reported to have got different repetitive elements (REP, homologous results from infective ERIC, DR, BOX or rep-PCR) in gram Casuarina sp. regardless of restriction +ve microbe genomes have also been enzyme used. Subsequently other opted for characterization of Frankia REVIEW OF LITERATURE 35 (Murry et al., 1997; Jeong, 2001). with distinct host range (Normand et Characterization of Frankia has also a/., 2007) Cluster 1 also known as · been done using antibiotic resistant "Alnus strains", include strains that can markers (Tisa et a/., 1999), pigment nodulate plants of Fagales, Betulaceae production and isozyme polymorphism and Myricaceae (Normand eta/., 1996), (lgual et al., 2001). and a subclade (within cluster I) of

Some recent works on Frankia "Casuarina strains" with narrow host taxonomy and diversity generating range that can nodulate Casuarina and BOX-PCR fingerprints targeting the Allocasuarina, (both species from conserved region of the 16S rRNA, Casuarinaceae), under natural using rep-PCR technique with BOX conditions (Benson et a/., 2004). primer on six Ceanothus sp. have been Cluster 2 comprises "Rosaceous carried out by Murry et al. (1997) to strains", which are not isolated in show Ceanothus nodules and culture and only infect five members of Elaeagnus infective Frankia share Fagales and Rosales comprising common ancestors. Coriariaceae, Datiscaceae, Rosaceae and Ceanothus of the Rhamnaceae Jose et al. (2003) conducted PCR of 16 (Benson et al., 2004; Vanden et a/., Frankia isolates using two different 2004 ). Cluster 3 comprises "Elaeagnus primers. (a) DNA fingerprints using strains". They form effective nodules DRIR primer through rep-PCR, which on members of the five families in the showed strain specific banding pattern Fagales and Rosales, which include and (b) RAPD fingerprints using 879F Myricaceae, Rhamnaceae, (16S rDNA sequence of E. coli). Here Elaeagnaceae and Gymnostoma of the it showed similar banding patterns with Casuarinaceae (Benson et a/., 2004 ). strain Ccl3 and UGL 020603 (isolated strain of different Casuarina sp. of Gtari et a/. (2010) are of the opinion different geographical origin), that Frankia strains based on 16S indicating that it may help in rRNA, nifH and gin genes (ITS identifying Frankia genomes at species region), cultivability, morphology and or sub-species level. infectivity, has been placed in four clusters. The classification put forward According to the studies conducted, here IS almost similar to the Frankia has been divided into three classification mentioned above, except clusters with members of each clusters REVIEW OF LITERATURE

Frankia placed in cluster II, which Frankia are nitrogen fixing, have not been isolated in pure culture, filamentous, sporulating, heterotropic, despite of many attempts and are gram positive bacteria of the order considered as 'obligate symbionts. Actinomycetales. These Such atypical Frankia strains m microorganisms have symbiotic association with Ceanothus, Coriaria, association with the roots of different Datisca, and Purshia are incorporated dicotyledonous angiosperms and some in Cluster 4. are free living actinomycetes in the soil

All approaches by different workers too (Wall, 2000). This ecological have not been made with an objective significance has made this to classify Frankia species. Some microorganism one of the important studies have been made to study the subjects for the researchers. Frankia strains from limited Frankia inhabit in three different geographic area or from single genus niches: the root nodule, the rhizosphere or species (Benson et al., 1984; Bloom and the soil. Based on characters like et al., 1989; Gardes and Lalonde, (i) ability to nodulate, (b) ability to fix 1987). Gtari et al. (2010) after carrying atmospheric nitrogen, (c) unique out 16S-23S rRNA ITS sequence from morphological properties, bearing 53 Frankia strains is of the opinion that sporangia and vesicles, (d) presence of this procedure is not useful to assign 2-0-methyl-D-mannose sugar (Mort et Frankia strains to their respective al., 1983), (e) presence of type III cell cluster or host because of less wall (meso-diarninopimelic acid, variability of Frankia genes among the glutamic acid, alanine, glucosarnine strains and phylogenetic relations are and muramic acid) (Lechevalier, 1994; sometimes ambiguous with nearest Myrold, 1994) and (f) high G+C% (68- matching strains. 72%) (Lechevalier, 1994; Gtari et al.,

Present! y, there is no universal Frankia 2010), Frankia has been uniquely taxonomy or any compilation on this placed in this genus. subject. Analysis of RNA nucleotide 2.9.1 Morphology and anatomy sequence 16S rRNA has been widely Actinorhizal nodules may have discrete accepted in Frankia systematics. branched lobes or be compact or may 2.9.0 FRANK/A-THE form nodule roots as in the case of MICROSYMBIONT Casuarina and Myrica (Berry and REVIEW OF LITERATURE 37 Sunell, 1990). It has been understood root hairs as m case of Alnus, that the host (actinorhizal) plant plays a Casuarina, Comptonia, Myrica, etc. significant role in modification of (Callaham et al., 1979; Berry and Frankia morphology (Berg, 1999; Torrey, 1983). Here, the bacterial Jeong and Myrold, 2003). Unlike hypha enters the root anatomy via root Rhizobium, Frankia induced root hairs (Torrey, 1976) and infection nodules are perennial and have proceeds intracellularly in the root different morphology and anatomy. cortex where Frankia endosymbiont is Anatomically legume root nodules are formed with vesicles and hypha! tip central and within endodermis with (Akkermans et al., 1989). tightly packed cortical cells, whereas in Simultaneously, there are cell divisions actinorhizal root the vascular tissue is in hypodermis and cortex producing centrally located to the infected cells wall like material composed of pectin and the nodule meristem is located at and hemicellulose, which is mixed with the distal tip of the nodule (Benson and intense branching of Frankia hyphae Silvester, 1993; Wall, 2000). within the cell (Berg, 1990) forming a

2.10.0 SYMBIOTIC INTERACTION­ small protuberance called prenodule A PROCESS (Callaham et al., 1979). Prenodules are infected with Frankia. Unlike The morphological steps of Rhizobium, nodule primordium m actinorhizal nodule development have actinorhizal plants is formed on been described in several excellent pericycle as a result of mitotic division, reviews (Newcomb and Wood, 1987; and not in cortex. Therefore, prenodule Berry and Sunnell, 1990; Wall, 2000). in the cortex infects primordium cells Process of symbiotic association in the pericycle to form a nodule. among these two genera has also attracted interests among many Intercellular penetration by Frankia workers. Depending upon the host hyphae takes place bypassing the root plants, two modes of infection of hairs that reaches direct! y to the cortex actinorhizal plants, after local through middle lamella between the colonization by Frankia have been epidermal cells, e.g. Ceanothus, described: intercellular and Elaeagnus, Hippophae, Shepherdia, intracellular. etc. (Miller and Baker, 1985; Liu and Berry, 1991; Huss-Danell, 1997). In Intracellular infection via deformed REVIEW OF LITERATURE

this case, there is no division in the support of Pseudomonas cepacia, cortical cell leading to prenodule whose presence may not be formation, instead cortical cells secrete compulsory in the process. There are electron-dense material rich in pectin also reports of signal exchange and proteins into the intercellular between Frankia and the host plant spaces, where the hyphae grow before infection initiates (van et al., (Racette and Torrey, 1989). As a result 1997; Ceremonie et al., 1999). of hypha! infection and cell division, However, no such active plant or the nodule primordium is formed similarly Frankia has been identified (Obertello as above in pericycle and plant cells et al., 2003).

gets infected by Frankia in the cortex, Apart from purification of hemoglobin developing to a nodule for further (Fleming et al., 1987) and isolation of infections (Miller and Baker, 1985). nodule-specific cysteine proteinase Mature actinorhizal nodules have eDNA (Goetting-Minseky and Mullin, multiple lobes, which consists of 1994 ), very little is understood about Frankia in cortical cells and central the molecular aspect of symbiotic vascular tissue. Interestingly, even in association between Frankia and the Parasponia-Rhizobium association plant. Recently it has been found that a prenodule formation takes place during set of actinorhizal nodulin genes are infection procedure (Lancelle and activated during differentiation of Torrey, 1984) and the nodules formed actinorhizal root nodules (Mullin and are also modified lateral root Dobritsa, 1996). Obertello et al. (2003) (Obertello et al., 2003) like defined two types of actinorhizal actinorhizal root nodules (Laplaze et nodulin genes based on their al., 2000). expression and pattern, by homology to

During the process of infection, legume nodulins. They are early Frankia sometimes takes the support of nodulin genes (involved in nodule soil bacteria to deform host roots. This organogenesis or plant infection) and idea was supported by Berry and late nodulin genes (involved m Torrey (1983) and also Knowlton and metabolism for nodules functioning). Dawson (1983) by mentioning that The type of infection and the form of Alnus rubra roots deform quickly and vesicles (spherical-Alnus, Hippophae, increase the rate of nodulation with the etc., mono-septate elliptical- REVIEW OF LITERATURE 39 Cercocarpus, Dryas sp., etc. and club Burggraaf et al., 1981). During or pear shaped Myrica, Casuarina, isolation various techniques were Comptonia, etc.) are detennined by the applied and it was found that Frankia host plant, and not by the are slow growing, heterotrophic, microsymbiont (Akkermans et al., aerobic organisms (Benson and 1989; Newcomb and Wood, 1987). Silvester, 1993).

2.1 1.0 ISOLATION AND Improvements in the culture media for STRUCTURE OF FRANKIA IN PURE in vitro studies of Frankia, from the CULTURE past yeast extract media to present

Pommer in the year 1959 reported first improvised composition of nutrient in vitro isolation of Frankia from the media, have now made the task easier roots of Alnus, which unfortunately got and endophytes also grow much faster lost, but his descriptions on the isolate (Lalonde and Calvert, 1979). Now matched with the present day Frankia successful isolation of Frankia from strain-HFPCpiL However, almost after almost all actinorhizal plants, except 19 years, first successful isolation of Ceuthostoma, Kentrothamnus, Frankia from the root nodules Chamaebatia, Dryas (Benson and Comptonia peregrine was reported by Silvester, 1993) and Adolphia (Huss­ Torrey and his collogues (Callaham et Danell, 1997) have been reported. Use al., 1978), which helped in of flavonoids (quercetin) in the culture understanding Frankia more closely media to control fungal contamination than before (Quispel, 1990). of Casuarina isolates have also been reported as a new measure of faster and Stowers (1987) reviewed successful contaminant free method of isolation of isolation techniques of Frankia in pure Frankia (Sayed and Wheeler, 1999). culture using different techniques, like serial dilution (Diem et al., 1982), In pure culture, Frankia behaves as micro-dissection (Berry and Torrey, microaerophilic and mesophilic organism and grows densely with 1979), Os04 surface sterilisation (Normand and Lalonde, 1982), filter branched and septate hyphae, exclusion (Benson, 1982; Weber et al., multilocular sporangia developing 1988), and sucrose density tenninally or at intercalary position of fractionation (Baker and O'Keefe, the hyphae and vesicles (Burggraff and Shipton, 1982; Newcomb and Wood 1984; Baker and Torrey, 1979; ' REVIEW OF LITERATURE 40 1987). 2.11.2 The sporangia

2.11.1 The hyphae: Sporangia are multilocular structures

Microscopic observations reveal that produced by all Frankia strains Frankia hyphae, with varying size of (Simonet et al., 1994).Sporangium 0.5 to 1.5 jlm, are mass of anastomose consists of spores, which are the (fuse) and branched mycelia to form a reproductive structures of Frankia dense mat. The cell walls of these (Krumholz et al., 2003). Each organisms have been found to be sporangium contains several hundred composed of electron-dense materials refractile infective spores that are 1000 in two layers: the base layer and the times effective in nodule production outer layer (Baker et al., 1980; Benson than equal volume of hyphae (Burleigh and Silvester, 1993). and Torrey, 1990). These spores are released from sporangia on maturity Reports also suggest that small round (Callaham et al., 1978) and are to spherical or rosary shaped, probably presumed that they help in dispersal glycogen and lipid granules, are and survival of Frankia but not much present in the hyphal cells (Benson and has been reported in this regard Silvester, 1993). Ribosomes and (Krumholz, et al., 2003). Inclusion of polyribosomes approximately of 300 2flg/ml of ampicillin (antibiotic) in the nm are present in the hyphal cells. culture medium increases sporulation Large number of circular shaped (in (Ganesh, 1993). cross section) cytoplasmic tubules (average 45 nm in diameter), Based on production of spores, two underlining the cell septum and outside types of root nodule were distinguished of the cell wall are also visible in the -one as spore positive (Sp+ ), periphery of hyphae cytoplasm comprising organisms that sporulate in (Lancelle et al., 1985). Newcomb et al. the root nodule, and the other as spore (1979) frrst reported an extracellular negative (Sp-), that sporulates in pure multilayered hyphae in a free living F. culture but does not form spores inside alni (HFPCpii ), but this envelop, the root nodule (van Dijk, 1978). All which is usually present in vesicles, Frankia strains isolated in vitro, till needs to be further studied and is a date, have genetic capability to produce subject of debate (Benson and spores at different levels, even if they Silvester, 1993). have been isolated from Sp- nodules. REVIEW OF LITERATURE 41 But all the isolates when re-inoculated suggest that 9 out of 25 genera of back to their hosts could not actinorhizal plants comprising six differentiate sporangia in planta (root families (Betulaceae, Myricaceae, nodules) and thus defined as Sp- strains Rhamnaceae, Elaeagnaceae, (Torrey, 1987; Simonet et al., 1994). A Casuarinaceae and Rosaceae) have typical Sp+ strain has not been isolated Sp+ nodules. Hippophae salicifolia too successfully (Simonet et al., 1994) but is associated with Sp+ Frankia strains. there are two reports on successful The morphological division of both the germination of spores of Frankia in the strains (Sp+ and Sp-) were further laboratory (Camet al., 1997). characterised at the molecular level Isolation of Frankia strains from Sp+ (Simonet et al., 1994; Wall, 2000). nodules was more difficult than Sp­ Simonet et al. (1994), based on 16S nodules (Schwencke and Cam, 2001). rDNA sequences, suggested that these In some cases, Sp+ nodules led to the two Frankia strains isolated from isolation of Frankia strains but these Alnus were genetically distinct from putative strains failed to differentiate each other.

sporangia in the root nodules when re­ Sporangia are multilocular structures inoculated in different host species. So located terminally or at intercalary they were classified as Sp- strains. position of the hyphae (Newcomb et Following this two hypothesis were al., 1979). Each locule or segment in proposed: (i) the phenotypic change of the sporangia contains many spores, Sp- and Sp+ may have been caused due which can survive in the host as well as to mutation thereby inhibiting or soil devoid of host (Weber, 1986). stimulating sporulation in the root 2.11.3 The vesicles nodule, and (ii) Sp+ and Sp- strains co­ exists in a single nodule and at the time In pure culture, vesicles are small of isolation, Sp- strains outnumber Sp+ spherical, thick walled structures, strains as former grows better in pure surrounded by multilayered lipid culture (Schwencke and Cam, 2001). envelope that arises from hyphae on a small stalk (Harriot et al., 1991; Sporulation in Frankia is not Gomaa et al.,2008), whereas in planta determined by the host rather it is their shapes vary from spherical to determined by the genotype of the elliptical to club shaped (Wall, 2000). endophyte (Schwintzer, 1990). Reports REVIEW OF LITERATURE 42

Vesicles are either produced when changes (Schwintzer et al., 1982; there is marginal or no concentration of Wheeler et al., 1983). During nitrogen in the medium or nitrogen senescence of vesicles, septum enriched medium where the nitrogen becomes irregular thereby ceasmg compound cannot be degraded to nitrogenase activity and disappearance ammonia (Zhang and Benson, 1992). of cytoplasm. It was also reported that During the formation of vesicles, the some vesicles rejuvenate to produce hyphal tip or side branches swell up to hyphae in vitro (Schultz and Benson, form pre-vesicles (1.5-2.0 f.!m), which 1989). may be non-septate (Wall, 2000) or 2.12.0 GENETIC DIVERSITY OF septate near the base and appears phase FRANK/A dark during examination with phase As mentioned before, actinorhizal optics (Newcomb and Wood, 1987). plants in symbiotic association with Finally these pre-vesicles mature to Frankia represent mostly perennial, vesicles (2.0-4.0 f.im) and are phase dicotyledonous trees or shrubs, except bright. Birefringence was observed Datisca (Jeong and Myrold, 2003) and under Polaroid light (Torrey and are globally distributed, except Callaham, 1982). From the experiment Antarctica (Simonet et al. 1999). They conducted by them using elegant freeze are being studied since 1829 fracture techniques, they concluded the (Schwencke and Caru, 2001). Along presence of a highly structured with the diversity of the host, there laminated layer, to be lipid monolayer. exists the diversity of the These layers were totally lost during microorganism. normal fixation procedures, leaving a In the present time, various reviews space around the vesicles called "void and reports makes us understand that area" (Benson and Silvester, 1993). there is a high degree of genetic Reports also suggest that vesicles diversity among Frankia strains harbor nitrogenase in Frankia isolated from different niches, plant symbiosis. Studies have correlated species, of same host plant or from a onset of nitrogenase activity with single nodule of specific location or vesicle development in young nodules different geographical areas (Mian and Bond, 1978) and its (Cournoyer et al., 1993; Myrold, 1994; appearance along with seasonal Normand et al., 1996; Clawson et al., REVIEW OF LITERATURE 43

1997; Clawson et al., 1999). plants (Hahn et a/., 1989; Honerlage et al., 1994; Benson et al., 1996; Lumini Classification (Becking, 1970) and phylogenetic studies (Lechevalier, and Bosco, 1996; Rouvier eta/., 1996; 1994) also aided in analysing the Normand et al., 1996; Clawson and diversity of Frankia. Global Benson, 1998; Benson and Dawson, distribution and adaptation of 2007; Sen eta/., 2008). Some works on actinorhizal plants in different genetic diversity of H. salicifo/ia has environmental stress (Tang et al., been done in India; however, no 2003), climatic and nutritional detailed work has been taken up in this conditions have led to the diversity of genus of Lachen Valley of Sikkim organisms. First isolation of Frankia Himalayas. strain in 1978 opened up even more 2.13.0 PROBLEM options for the study of diversity. Since IDENTIFICATION

then number of Frankia strains have To combat the harsh climatic been isolated from several actinorhizal conditions and extreme winters in plants from different geographical Lachen, North Sikkim, most of the origins, which have helped m people residing there rely on forest understanding its host specificity, products to meet up their daily metabolism, taxonomy and genetics consumptions in terms of fuel and (Benson and Silvester, 1993). fodder. Apart from private agricultural DNA isolation directly from the root land, which may be insufficient for nodule, amplification through PCR agriculture, forests are also used for using appropriate primers and intercropping of different agricultural sequencing has made it possible to and horticultural products like analyze uncultured Frankia strains. cardamom and potatoes. Therefore, Construction of phylogenetic trees with the increase in population these using related 16S-23S rRNA gene trends may not be economically and sequences from the GenBank and ecologically viable every year and in comparing with the isolated sequence the same locality for the local people using bioinformatics tools and due to ecological degradation. Besides appropriate software have been carried that, Sikkim is an organic state, so out in the past for studying diversity of there is a prohibition in the use of different endosymbiont of actinorhizal chemical fertilizers. Therefore, REVIEW OF LITERATURE 44 sustainable environmental and resource From the silvicultural point of v1ew conservation planning along with also, genetic differentiation of sea utilization of nitrogen natural! y has buckthorn plant needs to be understood become one the rnaj or target as to protect its genetic diversity and survival strategies for the inhabitants of restoration in the forests. Though there these high altitude areas. are some reports on genetic diversity

Nitrogen being one of the important and genetic differentiation of sea components of high altitude ecosystem, buckthorn (Yao and Tigerstedt, 1993; choice of naturally growing Bartish et al., 1999; Sun et al., 2006), actinorhizal plant species like H. many have contributed mainly on salicifolia with proper management origin, evolution and phylogeny of the and further scientific study can satisfy genera (Bartish et al., 2000 and 2006; the long-term conservation and Sun et al., 2002; Sheng et al., 2006). sustainable needs of the local However, till date no major record of population of this arid desert. Efforts genetic diversity of H. salicifolia and should be made on scientific study of its rnicrosymbiont Frankia have been nitrogen fixing symbiotic association reported. Therefore, knowledge on of this plant. H. salicifolia has genetic diversity and population remained virgin m this regard. genetics of this genus along with However, some research on symbiotic relationship and diversity of sporulation, host specificity, its endophyte-the Frankia, still further needs to be worked out especial! y in biochemistry, physiology and phylogeny using molecular techniques this region. may further bring light on the "If we consider Jawaharlal Nehru's idea, that endophyte and the host, for its future actual world situation is the result of the European domination in ancient social use. Choice of strain with maximum economical and military development; maybe nitrogenase activity shall help in sea buckthorn could have had another position developing a better symbiont for more in the history ofplant utilisation"

nitrogen supply to the plant and .. 00 00 .. Angel Proorocu generation of biomass. Chapter 3 Materials and Methods

3.1.0 THE STUDY AREA beautiful snow covered mountain

Sikkim, the 22"d tiny Himalayan State range. Available rainfall data shows of India is located between 28° 07' 48" that the mean annual rainfall of tbe and 27° 04' 46" N and 88° 00' 58" and state ranges from 82 mm to 3494 mm 88° 55' 25" E (Anonymous 1998, (http://sikkim.nic.in/sws/ 1982). It is situated on tbe flanks of tbe sikk_geo.html) and is the drainage Eastern Himalayas, and is bordered by basin of river Teesta. Variation in tbe Tibetan plateau in tbe north, Nepal altitude from south to north in less than in the west, Bhutan in the east and 100 km has resulted in micro-climatic Darjeeling district of West Bengal changes. Thus the different climatic (India) in the south (fig. 3.1) conditions encountered in Sikkim are sub-tropical (1000ft.-4500ft. amsl), The State constitutes a mountainous temperate (4500-8,900ft. amsl), sub­ terrain spread over 7,096 sq km alpine (8900-12,900ft. amsl) and alpine (Anonymous, 1998) is quit known (above 12,900ft. amsl). landforms of resplendent floral and faunal aggregation. The state However, tbe study area (fig. 3.1 and predominantly is mountainous virtually fig. 3.2), is popularly known as Lachen with no plain land. The altitudes above Valley, falls in tbe north district of mean sea level (amsl) varies from Sikkim, which lies between 27° 46' 31" 1000ft. in the south Sikkim to 25,800ft. and 27° 42' 30" north latitude and 88° in tbe north. The world's third highest 30'00" and 88° 35" 00" east longitude mountain Mt. Khangchendzonga and approximately 130 km away from (28,373 .40ft.) adorns tbe state with its the state capital, Gangtok. MATERIALS AND METHODS

The sea buckthorn growing in Lachen selected from the image and verified on Chu (river) watershed broadly includes the ground through field visit. These Yunga, Dozam, Zema I, Zema II, ground-truthing points were used to Lachen and Chaten. Yunga in the spatially resample the images using a North and Chaten in the South bind the nearest neighbour algorithm, which study area, approximately 27 km away takes the value of the pixel in the input from the nearest sub-division town of image that is closest to the computed Tchungthang. The bank of river Teesta co-ordinate. This method is fast and from Dozam up to Chaten has been does not alter the original pixel values. selected for the present study. Lachen The transformation had a root mean valley constitutes the block of the study square (RMS) error of between 0.4 and area. 0.7 pixel, indicating that the image

3.2.0 SURVEY OF STUDY AREA rectification was accurate to within one pixel. After registration the TIF Extensive survey of the study area has imagery is converted into image format been done for three consecutive years imagery which describes all the keeping in mind the objective of the information of the image like scale, work. resolution, projection etc. The geo-coded, 1:50000 scale Indian The density of the plants was estimated Remote Sensing (IRS) 1D LISS III by eyes and in some of the doubtful False Colour Composite (FCC), areas in the satellite imagery, during January 2002 data along with Survey interpretations the data were correlated of India (SO I) 1:50000 scale topo­ with the ground truth data. Using the sheets (78A/5, 78A/6, 78A/9 and remote sensing satellite data, the GIS 78A/l0) were used for the general land software and ground truthing, different classification and mapping with the ecological maps were created of the help of ERDAS ver. 9.0 imaging and study area viz. land use and land cover Arc map ver. 8.3 software. The images map, slope map, aspect map and were geometrically corrected and geo­ elevation map (may refer at fig. 4.1) to coded to the Universal Transverse determine the total actual ecological Mercator (UTM) co-ordinate system, conditions of the Hippophae salicifolia using 1:50000 scale approximately 20 growing area ofLachen Valley. evenly distributed ground control points (ground truthing points) were MATERIALS AND METHODS 47

1 KALEP 2 TALAM 3 lHONGU 4 ZEMA 5 LAC HEN 6 RHABOM

Figure 3.1: The study area showing the map of India with a small spot depicting the map of Sikkim, which is enlarged. The shaded area in the map of Sikkim is the study area, which is further enlarged to a bigger watershed area starting at the top from Kalep to Rabong. The sea buckthorn (Hippophae salicifolia.) plant is very scantly found in these areas and densely located in the areas of Lachen, Zema and its adjoining areas

3.3.0 E N V I R 0 N M E N T A L salicifolia. For this study, directions STUDIES from different taxonomists including

The literature regarding sampling Prof. A. P. Das, University of North intensity was not found to study H. Bengal, Dist. Darjeeling (India) were MATERIALS AND METHODS N A

Legend • Quadrate D Location - Hippophae I?: ':,;,I Dense Evergreen For~~??'r.~, D Open Evergreen For·esld:::-~

- River -water Channel ah~a,Y·~~"":"--.===:!"'"---!Kilometers 4 6 8 [!JYUNGA IIJDOZAM (l]ZEMA I!)ZEMA I illZEMA II (!]LACHEN IIJCHATEN Figure 3.2: Land cover map of the study site using geo-coded Indian Remote Sensing LISS III satel­ lite data (1:50000 scale) and GIS ERDAS solicited. these areas were namely, Zema ill,

A total of 20 different quadrates Zema ll, Zema ll+, Zema ITA, Zema throughout were laid out to study liB, Zema llC, Zema liD, Below different geophysico-chemical Dozam, Dozam+, Zema lA, Zema m, parameters. The plant morphology and Puchi, Chako River (first river from important taxonomic data of the genus Lachen towards south-east, below the were recorded in a prescribed format road), below Chaten, Chaten, above (fig.3.3). Chaten, Lachen River (Below Lachen towards East), Lachen River (other side The quadrates were named as per the of the river towards west), below socioeconomic information from the Lachen (other side of the river) and local people. The quadrates laid out in Chako River (first river from Lachen MATERIALS AND METHODS 49

SAMPLE NO: ...... DATE: ...... : ...... GENERAL INFORl\'L<\. TION: (i) Collection site: ...... (ii) Scientific name: ...... (iii) Nodules: Present/Absent. (iv) Nodules collected: Yes/No. (v) Root collected: Yes/No. (vi) Seeds collected: Yes/No. (vii) Soil Collected: Yes/No. (viii) Twigs collected: Yes/No Hard wood/Soft wood. (ix) Leaves.collected: Yes/No Male plani/Female plant. (x) Fruits collected: Yes/No. (xi) Barks collected: Yes/No Male plani/Female plant. (xii) Any other collections: ...... (xiii) Aerial temperature: ...... (xiv) Humidity: ...... A. HABIT AND HABITAT (a) Tree/Shmb/Herb (b) Flowering period: (c) Rainfall season: (d) Altitude: (e) Latitude: ...... Longitude: ...... (I) Topography: (g) Aspect: (h) Topogmphy: Swamp/Hilly!Riverine!Nonriverinefmounlain/others. (i) Vegetation type: Natural forests/Road side/social forests/other (j) Management: Cutting!Buming/Natural!Prcserved/plantation/others. (k) Associated species: (I) Site cover: Bare/Up to 20%nl-40%/41- 60%/61-80%/81- 100% B. SOIL TYPE: (i) Soil texture: Sandy/Loumy/Clayey!Rockcy/others. (ii) Soil pH: (iii) Soill temperature (iv) Soilmoisture: (v) Soil colour: Red/ Ycllow/Brown/R!ack/Grey (vi) Drainage: Flooded/Poorly drained/well drained/ C. NODULES (a) Location: Crown area/tap root/aerial root/main root. (b) Growth fonn: Clumped/scaltered

COLLECTED BY: NAME: ...... SIGNATURE.

Figure 3.3: Sample datasheet used for collection of gennplasm and recording field data.

towards south-east, above the road), Each quadrate of 20m x 20m for trees, which is collectively called the Lachen within which two Sm x Sm for shrubs Valley, under Tchungthang Sub­ and three lm x lm quadrates for herbs Division of North Sikkim (table 4.5; were laid out in different places Fig 3.1). One quadrate was studied for comprising riverside, torrents, each site. landslide areas and non-river side MATERIALS AND METHODS so areas. Different geo-physico-chernical Importance Value Index (lVI) of parameters like altitude, latitude, different species associated with longitude, aspect were recorded by Hippophae salicifolia following the

Global Positioning System (GPS) methodology of Coroi et a[. (2004). (Garrnin ETrex Vista H). Other data The parameters were calculated as such as aerial temperature and follows: humidity, were recorded using digital !.Abundance (A)=Total number of thermometer (Multi thermometer, CE individual species in all the make), huniidity meter (Model LR,; quadrates. Mignon AA, England make), soil pH 2.Density (D)=Tree density of and moisture were determined using on individuallha the spot tester soil pH and moisture 3.BA= Total basal area of each tree meter (Takemura, Japan make, model species!ha DM-15) and the soil temperature with soil thermometer standard field equipment. Soil quality, colour, topography, site cover, vegetation type, Circumference at Breast Height (CBH) in centimeters at the basal area of the tree species, management and species association were also studied in each of lmpcn-tmu:e IIalue !nder (IV"/)= HlJ +Hi'+ HJJom the 20 quadrates. The Importance Value Index (lVI) (i.e. Phyto-sociological characterization of Structural role) of each species was vegetation with reference to H. calculated by summing up the salicifolia (table 4.6) was prepared to observations of relative density (RD), study species richness and association relative frequency (RF) and relative of different species in the study area dominance (RDom). All the data and from the 20 nos. of quadrates. The sociability recorded were fed in a calculations of each tree species was computer as experimental attempts at converted to per hectare and placed in rapid analysis using Microsoft Excel the tabular form using GIS ERDAS software. software format of calculations. Calculations were made to study MATERIALS AND METHODS 51 3.4.0 R 0 0 T N 0 D U L E yards reflective) (iii) Canopy width in QUANTIFICATION feet using standard measuring tape on

Root nodule quantification of H. the ground, (iv) Tillering (number of salicifolia was carried out by randomly branches from the base), (v) average selecting 20 plants each ranging from 2 leaf width and pedicel length measured -7 years of varying height from riverine in centimetre from 100 leaves per plant (river-side) and non-riverine (non-river from different regions, (vi) average side) in different localities of the study inter branch distance measured in area. The vegetation around the plant inches per plant, using standard of interest was cleared and soil was measuring tape, were also recorded to dug to reach the nodules in the roots. In find out whether these factors had any some areas there were Alnus sp. impact on nodulation rate. All the data growing along with H. salicifolia in the were tabulated (table 4.8, 4.9a & 4.9b) same cluster, so care was taken to and graphically illustrated (fig. 4.4). avoid confusion and trace only the 3.5.0 GENDER Hippophae nodules. Parameters like (a) DIFFERENTIATION

Mean inter-nodular distance, in inches, Gender differentiation morphologically per plant using standard measuring could be differentiated at the flowering tape, (b) All nodules count per plant and budding stage. On the basis. of and, (c) Nodule weight per plant using floral and bud characters, gender could pan-top balance (Docbel, Brawn be distinguished. About 20 male and make), were taken into consideration female plants were randomly selected and recorded in the field. Other bio­ and tagged during the month of physical data like (i) Plant age, which December-January, April-May and was determined optically based on September-October so as to confliTll dendrochronology by counting annular the gender of the tree and also to see if rings of the lower branch of the species there were any cases of alternate or and corresponding its age with the irregular fruit bearing characteristics rings present in it following the process and other morphological features. of Oremus (1979). (ii) Plant height, 3.6.0 GERMPLASM COLLECTION determined by Laser range finder/ The germplasm for carrying out hypsometer model-400XLIXT/LH experiments based on the objectives of (Range-up to 400+yards passive 999 MATERIALS AND METHODS 52 this work was collected from various localities were stored in plastic bags locations and altitudes of Lachen separately containing moistened tissue valley based on the format at fig. 3.3. paper to maintain favorable water Ripe fruits were collected during the potential and kept in an ice-box with month of December, stored in a ice to slow down the growth of polythene bag and brought to the contaminants and limit tissue laboratory, after which it was dried to deterioration. Soil samples collected separate seeds with the dried fruit pulp for study of essential nutrients and for germination performance. nodules in plastic bags were brought to

Leaves and barks from both male and the laboratory and placed in deep female plants were also collected at the freeze at -20°C. nodules which, were same time for antioxidant studies. chosen for isolation purpose were kept Along with these hard wood and soft at room temperature as suggested by wood cuttings of 1-2 inches diameter Dr. Luis Tisa of UNH, USA and and 6-8 inches length were also wasting no time they were put into collected for propagation performance. media following different protocols as described later. Apart from study of nodulation rate, root nodules were also collected from 3.7.0 SOIL ANALYSIS various locations for isolation of Essential data like soil temperature, Frankia-DNA and Frankia m pure soil moisture, pH, aerial temperature, culture using different media humidity, altitude, aspect, nodulation, compositions. During collection of soil texture & color were studied from nodule samples, whitish or creamy the different quadrates of the study area coloured nodules were chosen because and noted down.

Frankia at this stage are reported to be The soil collected from different layers active. at a depth of 8-10 inches below the Percentage of nodulations were surface (after removing the topmost calculated by the following formula soil) were stored in a Jam bottle tightly (Raman & Elumalai, 1991) capped, labeled and brought down to the Tea Board, Government of India, Nwnber of plants with 110dules Percenlaoe (%)of 110dulation = umb f !ants b d X100 N era p o serve approved Soil Testing Laboratory All the nodules collected from different University of North Bengal, for chemical analysis using their standard MATERIALS AND METHODS 53 protocol. Different parameters of the m room temperature. With some soil like pH, Electronic Conductivity modifications to the process opted by (EC), Organic Carbon, Nitrogen, Rongsen, (1992) and Subedi and Potassium, Phosphorous, Sulphur, Adhikari (200 1), seeds were soaked Chloride, Silt, Clay, and Sand were overnight. The imbibed seeds were tested (table 4.7). then divided into three parts to see the

3.8.0 AGROTECHNIQUES germination performance in different soil based media (table 4.10). 3.8.1 Seed germination performance Germination performance using Frequent and extensive surveys on physiologically active substances (table occurrence of the plant and 4.11) and response of chemicals in morphological studies were carried out germination of sea buckthorn seeds in different seasons i.e. flowering were also studied (table 4.12). season (February-March), fruit The germination performance in ripening season (November-February) different soil based media was done in and vegetative growth season (April­ raised beds with different soil types/ July). In the month of June, the soil compositions like black, brown, specimens of the plant parts were also perlite and sandy soils without any collected from different areas and chemical or hormonal treatments. herbarium were prepared for further Timely watering and weeding was studies and records. Study on done to all the beds, and data like propagation of the plant was carried number of days for initial germination, out in the laboratory and hardening number of days · taken for 50% shed of Sikkim State Council of germination, number of days taken for Science and Technology, Gangtok, at total germination percentage and 4700ft. average growth in centimeter per Propagation of seeds was taken up month was recorded, from the day using freshly collected matured fruits when sprouting was observed in from the natural habitat in the month of individual beds. January-February. Juice was extracted Study of germination performance from the fruits, dried for 15-20 days, using physiologically active substances washed thoroughly in plain water, was carried out following Soyler and again dried for 2-3 days and stored dry Khawar (2007), with some MATERIALS AND METHODS 54 modifications. The already imbibed transported to the trial plot. Before seeds were treated separately with treatment, the cuttings were again cut different concentrations (600ppm, at the base to expose the fresh tissue 700ppm, 750ppm and 800ppm.) of and were pretreated overnight by

Gibberellic Acid (GA3) for 5 hours and dipping the lower portion of the wood grown in a petridish on Watman filter in the solutions bearing different paper No. I, labeled separately for each concentrations (table 4.!3) of IBA, set of treated seeds. A control (without NAA and Indole Acetic Acid (IAA). any treatment) was also placed just in Except the lower dipped part, rest distilled water to see the marked exposed or cut portions of the cuttings difference due to treatment on seeds. were waxed to prevent dehydration.

Apart from root and shoot growth in The treated cuttings were planted into centimeters, Critical Difference (CD) separate nursery bed of 0.5 x 0.5 sq. m was also calculated. within 15 em spacing (fig. 4.7). The The third kind of experiment (table soil composition was maintained as 4.12) on seed germination was carried sand: soil: compost at 70:20:10. out to see the response of chemicals in Watering was done as per the climatic germination of sea buckthorn seeds. In conditions and requirement. Twenty this case also the seeds were cuttings were used at a time for germinated in Petri dishes after treating treatment with each concentration of them with different concentrations of 50ppm, 55ppm & 60ppm. of IBA, chemicals like Sulphuric Acid (H2S04) 300ppm, 350ppm and 400ppm of NAA

(1.5N), GA3 (700ppm), Indole Butyric and 50ppm, lOOppm and 200ppm of Acid (IBA) (lppm), Napthelic Acetic IAA. The result was recorded and Acid (NAA) (5ppm) and control (only tabulated accordingly (table 4.!3). The distilled water). whole process was carried out in a

3.8.2 Propagation through cuttings controlled condition in a polyhouse.

Both hard wood and soft wood cuttings 3.8.3 Layering of the size of 21-28cm were taken Air layering was carried out in the from matured (approximately 4-6 month of April in 20 numbers of years old) trees from the natural approximately 4-6 years old tree orchard, bundled together, wrapped in branches (2-3 year old) mostly from a moistened muslin cloth and plants facing south east . aspects. MATERIALS AND METHODS 55 Layerages were prepared by covering Research Laboratory, Mumbai, India. the nodal portion of the branch using Aluminum chloride (AlCl3) was local moss and soil mixture wrapped obtained from SD fine Chemicals Ltd., by transparent polythene strips and Mumbai, India. tying the whole structure. It was left All chemicals and solvents were of untouched for three months and analytical grade. collected in the month of August. The 3.9.2 Plant material and extraction layerages were transferred to the trial plot (4 700ft.) for its propagation Both male and female barks and leaves performance. of Hippophae salicifolia were collected from different locations of the sacred 3.9.0 ANTIOXIDANT ANALYSIS forests of Lachen valley of North 3.9.1 Chemicals and reagents Sikkim during June 2010. The leaves Following chemicals were used for the and barks collected were dried in study of presence of antioxidant in shade. 10 g each samples were taken, different parts of Hippophae ground and extract solutions prepared salicifolia. using Soxhlet apparatus at boiling

2,2-diphenyl-1-picryl-hydrazyl temperature for 6 hours, with 80% (DPPH), Quercetin, Sodium Nitrite aqueous methanol, double distilled water (DDH20) and acetone (NaN02), Trichloroacetic acid (TCA), Ascorbic acid, Ferric chloride (FeCb), separately, at a ratio of 1:15 w/v plant Gallic acid were procured from HI material: solvent. All the extracts were Media Laboratories Pvt. Ltd, Mumbai, evaporated at a pressure of 50°C to India. make their weights constant. Later these extracts were individually Potassium di-hydrogen phosphate dissolved in DDH20 of desired (KH2P04), di-Potassium hydrogen concentration, before using for phosphate (K2HP04 ), Sodium experimental purpose. hydroxide (NaOH), Gallic acid, 3.9.3 Determination of plant extract Potassium ferricyanide (K2Fe(CN)6), yield Sodium carbonate (Na2C03), Hydrogen peroxide (H202) and Methanol were Based on dry weight, the yields of obtained from Merck, Mumbai, India. extracts were calculated using

Polin- Ciocalteau reagent from Sfsco following equation: MATERIALS AND METHODS s6

Yield (gj100gof dry plant material)= (W1 X100)/IV2 3.9.6 Antioxidant properties of Where, Wl=the weight of the extract extracts in vitro after the solvent evaporation and, 3.9.6.1 Free radical scavenging activity (DPPH method) W2=the weight of the dry plant material. Based on the scavenging effect of the

3.9.4 Determination of total phenolic stable DPPH free radical, anti-oxidant activity of all the standards and extracts content were determined following the The method of Singleton and Rossi methods of Hasan et al. (2006). (1965) using Folin-Ciocalteau reagent Spectrometric absorbance of each was opted to determine the total solution was determined at 517 nm soluble phenols in the extracts. Using after taking blank reading. The Thermo make UVI-Vis remaining DPPH scavenging activity in spectrophotometer, the absorbance of percent was calculated using the the blue color that developed was read formula: at 7 60 nm. Gallic acid was used as a standard. The concentration of total DPPH sf.Tlflenaing acti'uily (%) = ~ - A1 X100 Ao phenolic compounds in Gallic acid was Where A =the absorbance of the determined in 1-1g of Gallic acid 0 control and, A =the absorbance in the equivalent using an equation obtained 1 presence of the sample. To scavenge from the standard Gallic acid graph. 50% free radical, IC50 value 3.9.5. Determination of total concentration of the sample was flavonoid content required In this case quercetin was used as 3.9.6.2 Reducing power assay standard in mg QE. With some The method of Oyaizu (1986) was modifications, method opted by Goyal followed to determine the reducing et al. (2010) was opted for the total power of the extracts. The absorbance flavonoid contents of the extracts with was measured at 700 nm and compared Aluminum Chloride (AICh). Here the with standards. Indication of increased absorbance was recorded at 510 nm reducing power could be assessed by and all the experiments were carried increase in absorption value of the out in triplicates. reaction mixtures. MATERIALS AND METHODS 57 3.9.6.3 Scavenging of hydrogen thoroughly with running tap water to peroxide remove excess soil and organic debris.

. The scavenging ability of the extracts After cleaning for some time, the lobes in this experiment was determined were separated from nodules and following the methodology of Ruch et further cleaned by washing several al. (1989). The scavenging activity time with water and mild detergent like percentage was calculated using the Extran (E Merck, India Cat No. formula: MA02). The nodule lobes were further washed with sterile single and double • • • Abs (rontroO- Abs (standard) H101st:a11engms arlivily (%) = Abs (control) X100 distilled water many times and brought near the laminar air-flow. Frankia, Where, Abs (control) is the absorbance being slow growing organism, it was of the H20 2 (2 mM) as control and Abs necessary to surface sterilize the (standard) is the absorbance of the nodule lobes to overcome extract/standard. contamination in pure culture by other 3.9.7 Statistical analysis soil borne and fast growing nucro­ Experiments as mentioned above were organisms (Lechevalier, 1994).

conducted in triplicates. Results were 3.10.2 Surface sterilization calculated as the mean ± SD (standard Nodules were collected from the deviation) for each sample. Graph Pad rhizospheric region of the plant. Prism, Version 4.0 (Graph Pad Therefore, along with soil attached to it Software, San Diego, CA, USA) was many other microorganisms are likely used for statistical analysis; the to adhere on the nodule surface that correlation coefficient (R2) was may infect slow growing Frankia in determined for showing correlations'. pure culture at the later stage. To A significant difference was adjudged overcome this, different surface at a level of p < 0.05. sterilization procedures and chemicals 3.10.0 I S 0 L A T I 0 N OF were tried carefully. They are: ENDOPHYTE Nodules were treated with 0.1% 3.10.1 Washing Mercuric Chloride (HgCh) (E Merck Nodules were collected as mentioned India CatNo.l7524) for 7 Minutes and in germplasm collection section above washed 7-8 times with sterile double (3.6.0). Nodules were washed distilled water. Nodules were then MATERIALS AND METHODS 58 treated with 30% Hydrogen Peroxide thoroughly. They were then peeled off (H202) (E Merck India Cat No. in Laminar Air Flow (LAF) 1.07209 .0250) for 30 minutes and (Klenzaides make) using sterile forceps washed with double distilled water and needles. Further, nodules were several times. Nodules were now treated with 70% Ethyl Alcohol treated with 4% Sodium Hypochlorite (C2H50H) for 2 minute and washed solution (E Merck India Cat No. thoroughly for 7-8 times with sterile 618420) for 7 minutes and washed double distilled water. several times with sterile double All the above mentioned processes distilled water. At the end the lobes were carried out in the laminar air flow were treated with 70% Ethyl Alcohol (LAF) cabinet to avoid contaminations. (C2H50H) (E Merck Germany Cat No. All the sterilants were prepared using K29824783) for 2 minute and washed sterile double distilled water in LAF. thoroughly several times with sterile Since H20 2 is photosensitive, surface double distilled water (Bqse and Sen, sterilization using this chemical was 2006). carried out in dark.

Nodules were treated with 0.1% 3.10.3. Isolation of Frankia m liquid Mercuric Chloride (HgCh) for 2 culture minutes, washed 7-8 times followed by After proper surface sterilization of the treatment with 4% Sodium nodules, isolation was done using Hypochlorite solution for 7 minutes; following techniques: washed with sterile double distilled 3.10.3.1 Technique 1 water 7-8 times. Few nodule lobes were taken in a Nodules were treated with 30% sterilized mortar and pastel, crushed Hydrogen Peroxide (H20 2) for 30 along with little amount of sterile minutes, washed thoroughly followed medium and homogenate prepared. by treatment with 98% Ethyl Alcohol The nodule homogenate obtained was (C2H50H) for 2 minute and washed inoculated in culture flask/tubes thoroughly several times with sterile containing about 20m! of liquid media. double distilled water. 3.10.3.2 Technique 2 Thoroughly washed nodules were treated with 30% Hydrogen Peroxide Surface sterilized nodules were washed (H20 2) for 30 minutes and washed for 8-10 times with sterile double MATERIALS AND METHODS 59 distilled water and were aseptically under the microscope (Olympus CX41, transferred into the flask containing model U-DA 8Fl8837) to observe the liquid medium. The nodules were then subsurface sporangia or presence of crushed with sterile borosilicate glass vesicles. Vesicles and sporangia were rod and properly plugged. measured with the help of ocular (100

3.10.3.3 Technique 3 x=lj.!m) and stage meter (0.01 j.!m) (Erma, Tokyo, Japan). Photography of The properly washed, separated and vesicles and sporangia were done with sterilized nodules lobes were cut into the help of 10 mega pixel camera smaller sections and placed in the (Olympus). liquid medium. Further sub-culturing was done to 3.10.4 Media for isolation remove minor contaminations and to Among range of Frankia isolation get pure culture of the strain for other media, different media were tried to experiments. find out the best medium among all 3.11.2 Maintenance of culture (Media composition at Appendix 1). Active cultures were maintained by sub 3.11.0 POST ISOLATION TASK -culturing after every 2-5 months by 3.11.1 Incubation and sub-culture homogenizing the culture to break After the inoculation, the culture flasks apart the hyphae and promote rapid were sealed with Para film M sealer . growth, during transfer.

(GMBH, Germany, Cat No 7016 11) 3.11.3 Decontamination and were transferred to the BOD Though sterilization was tried to do incubator (Remi make, India) at 28°C. with perfection, still contaminants were The culture flasks were incubated for seen in the culture tubes due to the 50-60 days for initial development of slow growing nature of Frankia. Hence Frankia colony. Routine inspections different anti-microbial chemicals were were done to verify any contaminations tried. For example 500mg/ml of or growth in cultures. Cyclohexamide (Actidione, Sigma, The slow growing Frankia colony was USA. Cat. No. C-2609) was used for aseptically withdrawn from the flask, removing fungal contaminants. stained with Methylene Blue, mounted Actidione inhibits protein biosynthesis on a slide with Glycerin and observed m eukaryotic cell by binding 80S MATERIALS AND METHODS 60 ribosome. Frankia being Gram +ve illumination. bacteria, lOmg/ml of Nalidixic acid (1- 3.11.5 Plant infectivity test Ethyl -1, 4 dihydro-7-methyl-4oxo-1,8 Plant infectivity was done m ex situ -naphthydrine-3-carboxylic acid) condition. The seeds were sterile as (Sigma Chemical Co. USA. Cat. No. explained above and germinated m 3143) was used. For Frankia N2 free earthen pots containing sterile soil m medium like DPM was used and the hardening shed. All together there subsequently plating was done. were 100 pots containing single 3.11.4 Nodulation test seedlings per pot. After 30 days of In vitro infectivity experiment was intense care and watering, freshly conducted following the principles of collected root nodules were crushed Robert Koch (1843-1910). During this with sterile water and about 20ml of process, the dry seeds collected from the crushed solution were inoculated ripe fruits were washed thoroughly, per pot at the base of the plant so it surface sterilized with 30% H20 2 for reaches the root of the seedlings. The 10 minutes in LAF followed by plants were kept and allowed to grow washing thoroughly with double in a controlled condition of 24-28°C distilled water and put into .sterile with 80-90% humidity. bottles containing different 3.12.0. ISOLATION OF GENOMIC concentration of nitrogen . free DNA Hoagland's media (refer Appendix for 3.12.1 Isolation of genomic DNA from composition) solution (1/4, 1/8 and the root nodules: 1116) on a paper bridge, in a sterile The nodules were collected from all the condition. After a weeklong of 20 quadrates for the study of genetic incubation, 100111 of 45 days old pure diversity. Out of these, 11 sites with culture isolate of Frankia was different geographic, climatic and inoculated to the germinating seed with ecological conditions were chosen for some rooting to see the infectivity of isolation of Frankia DNA. The DNA Frankia to its host. Twenty H. isolated from the root nodules of these salicifolia seedlings were used in this sites were subjected to PCR process. They were allowed to grow in amplification and the products the growth room with controlled sequenced commercially along with conditions of 26°C with 11000 lux MATERIALS AND METHODS 61

PCR-RFLP as described below crushed m sterile refrigerated mortar

The nodules collected from each plant and pastel with liquid nitrogen. in different locality were numbered. Simultaneously, another technique was All these samples, after transporting also opted, where thoroughly washed from the study area to the University of nodule lobes were dipped in absolute North Bengal (laboratory), were ethanol (E Merck Germany Cat No. washed to remove excess soil and other K29824783) for 1.5 hrs to dehydrate; debris and stored in deep freeze at -20° dried for 15-20 min at room C for further use. temperature and crushed. But nodules

After cleaning, all these samples were crushed with liquid N2 gave desired taken for PCR based diversity studies. results. Considering various factors like The crushed nodules were transferred phenols, size of nodules, etc., method to 30ml pre-warmed (for 10 minutes at of Bajwa et al. (2005) was opted, with 65°C in water bath) Oakridge tubes, slight modifications. Isolation of DNA containing 5ml of DNA extraction from the root nodule was carried out buffer (Please refer Appendix for with following procedures: composition) with 1% PVP

Few nodule lobes (2g) was washed (Polyvinylpyrrolidone) (Sigma Cat No. thoroughly in tap water and later with PVP 40-500G) and 0.2% or 10 J.ll 2- distilled water. They were then dipped mercapto-ethanol (Sigma Cat No. in 2% activated charcoal (E Merck, M3148). The whole mixture (solution) India Cat No. 102183) and stored at 4° was again incubated in a water bath at C for overnight to get rid of phenolic 65°C for 1 hr. The solution was compound from the nodules. intermittently shaken and swirled carefully to ensure proper mixing for The nodule lobes were thoroughly reactions to take place properly. This washed with plain water and later with solution was carefully removed from double distilled water; they were the water bath after 1 hour and equal surface sterilized with 30% Hydrogen volume of Chloroform: Iso-amyl Peroxide (H20 2) for 15 min, washed alcohol (C: I) in the ratio of 24:1 with autoclaved double distilled water; (Merck Cat No. 6024450500 and dried under the normal room Merck Cat. No. 8189691000 temperature for about one hour and respectively) was added, mixed gently MATERIALS AND METHODS phenols mixed with DNA pellets); For all the samples reading was taken supernatant was transferred in fresh in triplicate. micro tubes. Each sample was mixed Ratio of OD26o/OD28onm was with 1/lOth volume of 3M sodium calculated to assess the purity of the acetate and double volume of chilled sample (OD ratios resulting between ethanol and stored overnight at -20°C 1.8-2.0 is reported to denote absorption for DNA to precipitate. of UV due to nucleic acids). A ratio The DNA samples were centrifuged at > 1.8 indicates the presence of proteins 7000Xg for 30 minutes at 4°C. The or other UV absorbers. A ratio <2.0 supernatant was discarded; the pellets indicates that the samples may be were washed with 80% ethanol and contaminated with chloroform or completely dried as described above. phenol. In either case (<1.8 or >2.0) re­ The completely dried pellets were precipitation of DNA using P:C:I dissolved in lOOf!l IX TE buffer and (25:24:1) and C:I (24:1) again, helped stored at -20°C for quantification. in purifying the impurities. It is

3.13.0 QUANTIFICATION considered that if sample OD at 260 nm reading is I, it has 50f!g/rnl DNA. DNA quantification as well as quality was done with UV vis­ The amount of DNA concentration of spectrophotometer (Themo Electro samples free of RNA was quantified in Corporation, England). Spectrometer nanogram ( ng) using the formula: for DNA measurement was set to the OD~l!ll_x1_00_..:0 (:..._dilu_tio_..:n f:..._act_or!...) X_SO.:.:Pfl:._ml DNA collCenlration (n9 m! ~ - wavelength 260nm (the wavelength of I 1000 light absorbed by DNA) and 280nm, as [1 f!g=1000 ng] under: Therefore, DNA Concentration or

600f!l TE (Tris-EDTA) Buffer (pH 8.0) Quantification (ng/rnl) =OD26o x 5000. was taken in a clean cuvette as blank 3.14.0 POLYMERASE CHAIN for zeroing. Six f!l of TE Buffer (pH REACTION AMPLIFICATION 8.0) was pipetted out from the cuvette, Polymerase Chain Reaction (PCR) was replaced by 06 f!l DNA sample to be performed following the methodology quantified, mixed thoroughly and of Bajwa et al. (2005) with some quantified at optical density (OD) at modifications. 260nm (OD26o) and 280nm (ODzso). MATERIALS AND METHODS

3.14.1 Primer: Life Sciences (P) Limited, #463 Pace

For studying genetic variation, a pair of City II, Sector-37, Gurgaon-122001, primers [FGPS 989ac Haryana, India (www.imperialls.com). (5#GGGGTCCGT AAGGGTC3#, Both the primers (forward and reverse) (Bosco et al., 1992) and FGPL were desalted having 25 nmol scales. 2054' (5#CCGGGTTTCCCCATTCG The forward primer G3#, Simonet et al., 1991)] were (5'GGGGTCCKTARGGGYY3') had initially chosen. The primers were 16 mer length with 68.8% GC content, kindly suggested by David Myrold, on molecular weight 4974g/mol and OD personal communication. However, 4.1. Its concentration was 23 .9nmol when we applied this primer pair for in (119!!1). Ultra-pure (pyrogen free) silico PCR amplification of Frankia water (Merck India) was mixed to the reference strains like ACN14a, Ccl3, stock making the final volume to 239!!1 EANl pee etc. the primers produced no (24pmoll!ll). The melting temperature amplification. We therefore modified (Tm) was 56.9°C.

the primers and ultimately designed a The reverse primer FGPL pair of degenerating primers, which 2054' (5'CCGGGTTTCCCCATTCGG worked for all the strains mentioned 3') had 18 mer length (Simonet et al., above. To make the primer pair a 1991). The GC content of it was 66.7% Frankia specific one, we tried to with molecular weight of 5443g/mol amplify genomes of several and OD 4.3. Its concentration was phylogenetically related actinomycetes 24.9nmol (136!!1). Like forward with no amplification. The primer, Ultra-pure (pyrogen free) water degeneration primers we designed are was mixed to the stock making the as follows final volume to 249!!1 (25pmoll!ll). The Forward primer melting temperature (Tm) was 60.5°C.

. 5'GGGGTCCKTARGGGYY3' Both the stocks were labeled properly Reverse primer and stored at -2o•c for further use. 5'CCGGGTTTCCCCATTCGG 3' Working solutions of 25!!1 of both the

3.14.2 The primer stock: primers were taken in a fresh I m1 micro tube, labeled properly and stored The newly designed primers were at -2o•c for frequent use. synthesized commercially by Imperial MATERIALS AND METHODS

35 CYCLES

3min. lmin. 2 min. 7 nUn.

lmin. Figure 3.4: Graphical representation of temperature and duration calibrated during performing PCR

3.14.3 Annealing temperature: 3.15.0 PCR REAGENTS:

In the present case annealing Bangalore GeNei TM DNA temperature (Tm) for forward and Amplification Kit (supplied in ice reverse primer was provided by the pack) with marker for 50 reactions (cat primer synthesizing company (Imperial No. 105548) was obtained and stored Life Sciences (P) Ltd., India), which at -20°C. Following materials were was 56.9C and 60.5C respectively. provided in the kit.

Therefore, annealing temperature was • 1. Taq DNA Polymerase calculated as follows: • 2. dNTPs . ~mafFDrHardPrmt r,o{Rmrse~ bmaling ~era!Jue= -5 • 3. lOX Assay Buffer with 15mM 2 MgCh 56.9HO.S) Annealing temperature = - 5 =53.7 ( 2 • 4. lOX Assay Buffer without MgCh

In the present experiment after various • 5. 25mM MgCh experimentations, annealing • 6. Control DNA and primers to temperature of 54 was standardized. perform 10 tests. Table 3.1: Concentration of different chemical constituents used for making PCR IlllX SI. Constituents Quantity for one Master mix for 12 No. sample (25"1) samples ("I)

I. Ultra-pure pyrogen free H20 19.7 236.4 2. lOX Assay Buffer with 15mM 2.5 30 MgCI, 3. Forward Primer 0.5 6.0 4. Reverse Primer 0.5 6.0

5. dNTPMix 0.5 6.0

6. Taq DNA Polymerase 0.3 (01 Unit) 3.6

7. TOTAL REACTION VOLUME 24fll 288"1 MATERIALS AND METHODS 66

• 7. DNA Marker 3.17.0 PCR MIX:

• 8. Instruction Manual Per sample total volume of PCR

Of the above reagents, only lOX Assay mix=25 fll, number of Buffer with 15mM MgCh, samples= II+ 1=12 (one control). Deoxyribonucleotide (dNTP) Mix, Taq The total volume of PCR master mix DNA polymerase and DNA marker (288f.ll) was divided into 12 equal parts were used during the experiment. i.e. 24fll each, in PCR tubes; lfll each

3.15.I Taq DNA polymerase and diluted DNA template from different assay buffer samples' was added to each 11 PCR tubes making the volume to 25fll. In Total of 0.6rnl of lOX Assay Buffer one PCR tube with mixture Ifll of bearing Lot No. I2030 (IOmM Tris­ Ultra-pure pyrogen free H20 was HCl (pH 8.8) with I5mM MgCb) was added to set it as a control for negative supplied in a small vial. This was control (please refer table 3.1). stored in -20°C. 3.18.0 PROGRAMME FOR PCR 3.15.2 Deoxyribonucleotide AMPLIFICATION: triphosphate (dNTP) mix PCR was performed in thermal cycler IOmM concentration of (Applied Biosystems make, 2720 Deoxyribonucleotide or dNTP nnx Thermal Cycler). Amplification (Lot No. 231030) was supplied m a programme consisted of one initial liquid form. The quantity of the cycle of denaturation at 94°C for 3min, solution in the vial was 2f.1mole with followed by denaturation temperature 0.2rnl volume. The solution was kept at 94°C for I minute, annealing along with all the working solutions at temperature at 54°C for 1 minute, -20°C. extension temperature at 72°C for 2 3.I6.0 DNA DILUTION: minutes and final extension 30-50ng/f.1l DNA concentration was temperature at 72°C for 7 minutes. taken for PCR work. I-2f.ll DNA was Total of 35 cycles were run for taken from the quantified sample and amplification of I6S-23S rRNA from serially diluted by adding IX TE buffer the root nodules of Hippophae to bring down the DNA concentration salicifolia of Sikkim (fig. 3.4). to 30-50ng/f.1l for PCR. MATERIALS AND METHODS

3.19.0 AGAROSE GEL GeNei ™) was set at 600mA and timer ELECTROPHORESIS: was set to 1.5 hours.

To check the efficiency, 1110 of PCR 3.20.0 GEL PHOTOGRAPHY: product was examined through The gel was placed over the electrophoresis on horizontal gel transilluminator (Bangalore GeNei ™) containing 1.5% (w/v) agarose (Sigma and photograph was taken using the cat No A9414) gel and ?f.!l Ethidium camera Canon power shot (SX 210 IS, bromide (3,8-Diamino-5-ethyl-6- 14.1 mega pixel, Japan make) for phenylphenanthridinium bromide) observation and further studies (fig. (Sigma cat No. El510). 4.12).

Agarose (!.5g) was dissolved in 100 3.21.0 RESTRICTION FRAGMENT ml of 0.5X TBE buffer and warmed LENGTH POLYMORPHISM until agarose powder got dissolved The PCR products were subjected to completely. After few minutes, ?fll of restriction digestion with 4 different EtBr was added to it and mixed gently, restriction digestion endonucleases, then it was poured over the gel casting viz., AZul, Haem, Mspl and HpaiT, all tray with comb inserted to it. After the 4-base cutters. The lists of gel was solidified, the comb was endonucleases (enzymes) with their removed and the gel was shifted to the cutting sites are given in the table 3.2. gel loading tray (Tarsons) containing The restriction digestion process was 0.5X TBE (Tris Borate EDTA) buffer. carried out in the following process. DNA (3f.ll) marker (ladder) of lOOObp supplied with the PCR kit (Bangalore Restriction digestion mixture of 20fll per sample was taken containing GeNei TM DNA Amplification Kit, Cat No. 105548) was loaded in the first following components for each sample: well of the gel. Simultaneously, 5f.!l of • Restriction enzyme buffer 2fll

PCR products were mixed with 2f.!l of • Pyrogen free water to make up the Bromophenol Blue solution volume. individually in all 12 (11 +!control) • Enzyme 5 units samples, mixed thoroughly and loaded • PCR product 5f.!l. gently in the individual wells of the gel. Negative control was loaded in the The restriction digestion mixture for last well. The power pack (Bangalore total of 11 PCR products were prepared MATERIALS AND METHODS 68 with basic combinations stated above, data matrix. A similarity matrix on the mixed thoroughly and distributed to 11 basis of band sharing was calculated parts. from the binary data using Dice

In each part 5f1l of PCR product was coefficient (Nei and Li, 1979). added and incubated at 37°C for I hour. Similarities were graphically expressed using the group average agglomerative The restriction digestion product was clustering to generate dendrogram. The separated in 2% ('NN) agarose gel POPGENE freeware (Yeh et al., 1997) with ethidium bromide solution (O.Sflg/ was used to partition genetic diversity ml) and run in IX TBE buffer (pH-8). among the 11 isolates of Frankia. The Gel loading dye 6X (5!11 was mixed same software was used to calculate with 20111 of restriction digestion genetic distances and similarities product. between populations and to draw a The samples were loaded and dendrogram based on Nei's (1972) electrophoresis was carried out at 50V genetic distances using the UPGMA to for 2hrs. show the genetic relations between the

The gel was visualised in UV different isolates. Graphical transilluminator (Genei make, Cat# representation of the results was done 107161) and photographed with canon on the basis of these studies. shot (21S, 14.1 mp) camera. DNA 3.22.0 NUCLEOTIDE marker (lkb ladder) (Genei, Cat# SEQUENCING:

105998) was used as molecular marker. All the amplified products of expected 3.21.1 PCR-RFLP data analysis: size were directly sent for sequencing

The markers were scored as diploid to Chromous Biotech Pvt. Ltd., #842, data. Each polymorphic band was II, Floor, Shankar Bhawan, A Block, regarded as a binary character and was Shankar Nagar, Bangalore-5600092. scored as 1 (presence) or 0 (absence) (www .chromous.com). for each sample and assembled in a Total of 100111 of PCR product for each

Restriction Cutting sites Temp. Units enzymes Table 3.2: Restric­ --"'?'==-----:-:::-:-:=-----::-=::-:::-----:5:---- tion enzymes show- A lui AG~CT 37°C H;,\m GG~CC 37°C S ing their respective '. cutting sites M:d>l., qCGG 37°C 5 Hpaii qcGG 37°C 5 MATERIALS AND METHODS sample along with 20j..tl each of for Biotechnology Information (NCBI) Forward and Reverse primers were web site (http://blast.ncbi.nlm.nih.gov/ pipetted into PCR tubes separately, Blast.cgi). labeled properly and sealed with 3.24.0 SEQUENCE SUBMISSION parafilm and sent for sequencing. TO GENE BANK:

3.23.0 SEQUENCE BLAST : Total of 22 partia l 16S rRNA All the 22 ( ll forward and ll reverse) sequences were submitted online to result sequences received from the E u ro pean M o l ecular B io lo gy Chromous Biotech, Bangalore, were Laborato ry (E MBL) nuc le o tide individually compared with the e q u e n c e d a t aba se equivalent sequences from a range of (http:\\.www.ebi.ac.uk/emb l) with other Frankia sp. present in sequence proper annotation and descriptions, banks using Basic Local Al ignment after registering to the web site (fig. Search Tool (BLAST) (Altschul et al. , 3.5). The procedure of the web site was 1990) obtained from National Centre fo llowed during the submission process

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atd.!l$. ffiW? b Figure 3.5: EMB L format used for submission of 16S rRNA sequence from root nodules of Hippo­ plwe salicifolia MATERIALS AND METHODS 70 diversity of Frankia strains obtained and sequences were submitted in from the root nodule isolates of H. PASTA format. The accessions salicifolia. Various 16S rRNA numbers of the sequences were sequences from other Frankia strains received and have been illustrated in along with that of closely related chapter IV. The data is also accessible actinomycetes members were also through NCBI GenBank. considered for phylogenetic tree 3.25.0 PHYLOGENETIC construction (fig 4.13). The bootstrap ANALYSIS: consensus tree inferred from 1000 The sequences were then subjected to replicates was taken to represent the phylogenetic analysis. For this multiple evolutionary history of the taxa alignment was carried out using the analyzed. The percentage of replicate software MEGA ver. 4 (Tamura et al., trees in which the associated taxa 2007). Neighbor joining method clustered together in the bootstrap test (Saitou and Nei, 1987) was used to (1000 replicates) is represented next to construct phylogenetic tree to infer the branches. evolutionary history and genetic Chapter 4 Results & Discussion

4.1.0 SOME SURVEY FACTS -5°C at upper ridges of Zema ill to

The study site with total area of 21 oc in the lower ridges of Chaten. 23427.62 ha is popularly known as All the maps used for ecological Lachen Valley and is surrounded by studies at fig. 4.1 (A, B, C and D) of sacred Pangal Reserve Forest. The the study area were prepared using middle and southern ridges (Lachen 1:50000 scale, geo-coded India Remote and Chaten) are moderately populated Sensing (IRS) 1D LISS ill False Zema lA, Zema ill, Zema II, Zema ill, Colour Composite (FCC), Survey of Puchi and Chalco have very less density India (SOl) 1: 50000 scale maps of population and rest of the area has (78A/5, 78A/6, 78A/9 and 78A/10) and temporary huts constructed for GIS using ERDAS ver. 9.0 and Arc seasonal agriculture. Due to severe map 8.3 software, along with ground winter and heavy monsoon, the area truthing, data collection and extensive sometimes remains cut off from the surveys as elucidated in 3.2.0 of rest of the world as there is only one chapter ill.

road considered to be the lifeline 4.1.1 Land use and land cover connecting Lachen from rest of the Lachen valley has a very interesting world. No information on authentic admixture of land use and land cover published climatic and topographical covering 7605.19 ha, including part of data of this area was available, but Pangal Reserve Forest. Here, during my regular visits for three years Hippophae salicifolia D. -Don (sea it was revealed that the temperature in buckthorn) plant is found growing ~- the study area ranges from minimum of natural! y in different areas in pure or RESULTS AND DISCUSSION 72

Table 4.1: Vegetation classification with area revealed from the land use and land cover map of the study area using GIS ERDAS. Vegetation Class Area (ha) Dense Evergreen Forest 7605.19 Open Evergreen Forest 6610.25 Scrub Forest 4466.69 Forest Blank 2183.19 Barren Rocky/ Stony area 728.94 River-water Channel area 509.88 Lakes 3.56 Grassland/Grazing land 323.50 Snow cover-Perennial 762.00 Hippophae-bamboo 19.83 Hippophae-broadleaf 60.90 Hippophae area 89.60 Hippophae-conifer 59.51 Hippophae-Alnus 4.59

Total 23427.62 mixed clusters. Table 4.1 and fig. 4.1A vegetation combinations like reveals different vegetation class and Hippophae-conifer, Hippophae-broad their areas in hectare and fig. 4.2A leaf, Hippophae-Alnus, Hippophae­ illustrates the percentage of different barnboo, etc. During digitization and vegetation and land cover of the study assignment of training sites to the IRS area. It is seen that major part of the satellite data through GIS (as stated study area is occupied by dense above) for creating map, only the areas evergreen forest (33%) which is due to which covered more than 1 pixel in the the part of Pangal Reserve Forest. The satellite data were accepted by the pie chart presents total area covered in system. This was due to very small percentage by different land use and patches of Hippophae growing areas land cover parameters through legends on the ground, which became 1pixel or shown in the map. In areas like forest less on the FCC data. In this work blank, barren rocky/stony areas, river­ Hippophae area was taken up manually water channel areas, lake, grass land through intensive survey, ground and snow cover (perennial), presence truthing and data interpretation, to of H. salicifolia was not observed. authenticate the ground data, which Beside these areas, H. salicifolia stands could not be verified only through could be observed in different image data. This was later on assigned RESULTS AND DISCUSSION 73

N N A A

t..gendf

~ l~&oldltlf .-,..,..,~..,..&lb • bJf«j(y ... • One _,_~~n:a . r-tw.t .

N A

Legends 0'· 90" \ I .J . 90' - 180•

. 180'- 270' 60 -75 . 27(1-360' I 1 75 · Above

Figure 4.1: Ecological maps of the study area u ing LRS 1D LISS Ill FCC, SOl topo -sheets (78N5, 78N6, 78N9 and 78Nl0) and GIS ERDAS ver. 9.0 and Arc map ver. 8.3 oflware. A: Land u e and land cover map, B: Elevation map. C: Aspect map and 0 : Slope map RESULTS AND DISCUSSION 74 Table 4.2: Area covered in ha by different elevation range revealed through GIS. Elevation range (ft.) Area (ha) 0-9000 4093.69 9000-13200 13274.90 13200-above 6058.99 Total 23427.58

• Oel>se EWfgreen Fcru • Open E""'V'een Fo

" 5now CC>Ire<- Perrero.t

·~~~-bamboo • ttppophee - broadleal • 0000-13200ft

IIH-hH••• w13200ft -- "H~e-eomer • "~· - Alnus

• 0-15% • G-OONE

11 91·180 SE

w181·270SN

11271·360 NW

Figure 4.2: Pie chart showing percentage of area covered by A: Different land use and land cover; B: Range of elevations; C: Different aspects and D: Range of slopes to GIS to produce the land use and land various models were created from cover map. ground samples usmg satellite data

Such ki nd of predictive land use and (FCC) fo llowed by GIS to extrapolate land cover maps covering vegetation across unsampled areas. Similar work have been used in remote sensing had been carried out by Roy et al. (200 I) on H. rhamnoides L. of Lahul applications to improve land cover and Spiti in Himachal Pradesh (India), classifications based on digital satellite data by Strahler (1981 ); Cibula and usi ng IRS IC LISS III FCC and GIS Arc-view software. Here, GIS was Niquist ( 1987). In these studies, RESULTS AND DISCUSSION 75 Table 4.3: Area covered in ha by different aspects revealed through GIS Aspect range Aspect Area (ha.) 0-90° NE 5241.0273 91°- 180° SE 6737.7199 181°-270° sw 6825.3649 271°-360° NW 4623.6112 Total 23427.723 limited to digitization for classifying separated by colours. From the map the satellite data. However, I have supported by ground data, it was found created GIS map with ecological that elevation of the study area ranged information of even inaccessible areas up to 13,200ft. The GPS aided ground through intensive surveys and data in the H. salicifolia growing in collection of field data using high different range of elevations are resolution satellite imageries and illustrated at table 4.2 compliments the Survey of India topo sheets, as stated data at table 4.5. Further, Pie chart at above. fig 4.2B and table 4.2 of the study area,

Such inventory map (Figure 4.1A) elaborates that maximum area covering providing ecological information 13274.90 ha of land falls at an proves to be a boon for the researchers, elevation range of 9000ft.-13200ft. planners, foresters and conservationists covering 57% of the total area. 17% towards identification and mapping of total area falls under altitudinal range important floral species and up to 9000ft. covering 4093.69 ha. and simultaneously applying noble the remaining 26% of the area falls conservation practices. under 13200ft. and above, covering 6058.99 ha land. Detailed observations 4.1.2 Elevation of data in pie chart at fig. 4.2B and The map at fig. 4.1B shows different altitudinal data in table 4.5 shows that height ranges of the study area most of the H. salicifolia plants grow at

Table 4.4: Area covered in ha by different slopes revealed through GIS Slope Range (%) Area (ha.) 0-15 2073.61 16-30 8808.19 31-45 11513.96 46-60 1028.37 Above 60 3.73 Total area 23427.86 RESULTS AND DISCUSSION an elevation rangmg from 9000- lands, grass lands or scrubby 13200ft. compared to 0-9000ft. Ground vegetation. SW aspect, which 1s reality also confirms that there was no opposite to NE aspect, received more H. salicifolia growing above 13200ft. sunlight and vegetation also flourished covering 26% of my study area better in these area. because the area was mostly covered Observation during survey and data by snow, grass lands and barren lands. collected by GPS navigation at table This may be due to the geological 4.5 also reveals that SE aspect had makeup of the Sikkim mountains healthy and taller tree stands of H. because reports by Rongsen, (1992), salicifolia compared to other aspects Rajchal (2009) and Singh et al., (1995) and tree stands at gully and drainage suggests that H. salicifolia grows areas receiving less sunlight. Out of 20 beyond this altitudinal region. quadrates, 9 slopes faced SE, 5 faced 4.1.3 Aspect SW, 4 faced NE and 1 each slope faced

The cardinal or aspect in the map at north and east. This clearly indicates fig. 4.1 C, of the study area, are based that H. salicifolia is a sun loving on angle measured clockwise due north species, which prefers growing mostly called azimuth (measured between 0- in sun facing SE aspects. My results 360"). Table 4.3 and fig. 4.2C corresponds to the reporting made by elucidates all the inter-cardinal Rajchal, (2009); Rongsen, (1992) and directions (aspects) and their area Singh and Awasthi (1995), where these covered, viz., north east (NE) covering authors have also advocated on growth 22%, south east (SE) and south west preference of sea buckthorn in open (SW) covering 29%, and north west sun facing aspects. Since such kind of (NW) covering 20% of the area. SE study is the first of its kind on H. and SW aspects cover equal area in the salicifolia of Sikkim. study site 4.1.4 Slope

Table 4.3 also reveals the individual Slopes are important ecological area coverage in hectares, for each component in identifying constraints aspect. From the ground survey it was and evaluating potential environmental observed that NW aspects received less impacts related to landform alteration sunlight than SE aspect and was mostly along with vegetation successions. Fig. covered with snow or were barren 4.10, table 4.4 and fig. 4.20 reflects RESULTS AND DISCUSSION 77 the fact that the study area consists of naturally and was most! y concentrated hilly slopes with virtually no plain in the river banks or riverine areas land. The table and the figures (RA) of Lachen River and torrential illustrates the slopes in the study area drainages connecting it. The plant was ranged from gentle (0-15%) to very also found to be growing in non-river steep slopes (above 60% ), individual side or non-riverine (NR), torrential classification of slope area in ha. and areas, slopes, village road side, grazing percentage of slope area covered. It forest, torrential banks and landslide was also observed that moderate slope areas. They were growing either in ranging from 16-30% to 31-45% small patches of pure populations or occupies maximum area of 11513.96 mixed with other vegetation (fig. and 8808.19 ha. covering 49% and 4.1A). It was also found that about 4-5 38% of the geographic location, year old plant was found growing in respectively. Above this percentage of the middle of the high current Lachen slopes, the terrain started becoming River forming a small island, which steeper and there was decrease in indicated the strength of its root and its vegetation. ability to hold the fragile slopes of sea

H. salicifolia growmg area covered buckthorn growing area. slopes ranging from 16-30% and 31- A very interesting admixture of two 45% slope landscape mostly with SE actinorhizal plant species was observed aspects. This was similar to the reports at an altitude of 7845ft. in quadrate (Q) of Rongsen (1992) and Singh (1998) 15. Here Alnus nepalensis population that Hippophae prefers growing in sun ended from the south and H. salicifolia facing aspect i.e., SE aspect. The slope population started towards north facing map gives an idea about the terrain SE aspect. Majority of H. salicifolia condition and also helps in future growing areas were fragile having new planning and management of the area soils with great risk of mass movement by different section of researchers. and flash floods. . Geo-physico-chemical studies of all the Some old H. salicifolia forests with tall quadrates carried out during survey trees were observed in Zema II+ (Q2) reflected interesting ecological facts of and below Lachen (Ql7, Q18 and Q19 the field (table 4.5). It was observed as referred in table 4.5). Other that H. salicifolia was growmg locations like Dozam, Zema liB and RESULTS AND DISCUSSION

Zema IID also had old stands, but stated above was found suitable habitat fewer in populations due to human for H. salicifolia in North Sikkim. interferences for fuel and fodder, From the socioeconomic interaction it slowly being replaced by naturally was understood that the sea buckthorn regenerated plants. This forest clusters was used in fencing of apple orchards, showed association with Arundinaria fodder for cattle, firewood and the fruit sp. Platt and Brantley (1997) suggested pulp was used in treating common that Arundinaria gigantea basically a problems like stomach ache, common species of river flood plains of the cold and fever. Ripe fruit juice of sea south, is fostered by burning regimes. buckthorn was used as nat1;1ral dyes for However, bamboo (Arundinaria sp.) colouring endemically woven clothes and Hippophae can be observed in and blankets. Need for fuel wood and many patches of the study area also construction has led people to cut down supports the idea of forest fires in the forest trees including sea buckthorn past. Results also indicated that H. plants. The plant also serves a good salicifolia is well associated with dietary supplement for the birds and Daphniphy/lum sp. Rhododendron sp. other animals (Fig 1.5). Tsuga sp. Populus sp., etc. (table 4.6), 4.1.5 Vegetation analysis which makes this species a major Understanding the forest structure is player in the ecological balance of this important to describe different fragile area. ecological processes and also to model The study area consisted of various the functioning and dynamics of forest admixtures of soil combinations (Elouard et al., 1997; Sukumar et al., (loamy, sandy, clayish, etc.), colours 1992). (brown to dark brown) and pH ranging In table 4.6 properties of vegetation from 4.5 to 6.9. The site cover of with reference to vegetation natural H. salicifolia orchard ranged composition and functional attributes from 20% to 61-80% (table 4.5). are expressed on species basis. The Furthermore, any area with density (D) measurement (number of geographical boundaries of 27°42'758" individual species present), relative to 2]D46'839"N and 88°31'041" to 88° dominance (Rdom) (largest species in 33 '639" E at an altitude ranging from terms of its presence), Frequency (F) 7845 to 10206ft. with pH and aspect Tabla 4.5: Field data compilation of the study area sholling different gee-physico- Tabla 4.5: continued. chemical parameters. Ql 02 OJ Q4 05 01 07 01 Q9 010 011 012 01l Q14 015 Q16 011 Q18 Qfl Q20 Lccation z.mam Zomal + z.ma I A lema DB Below Dmam + Dozam Zoma DD lema I C Z.ma I+ LDC>Iitln lema I lema I Puclii Chako Below Cllaten tbole Below Behlw Cllako Do zan (A) (B) (lema I) Rrver Chaen Chaten Lachen Lachen 1<'/tr Date amooa 3.1210!B amOOB amoo8 4.12.2008 4.1210JS 4.12m 4.12:lll08 4.1220118 5.12.2008 Date 5.122008 5.12.200 5.12100 5.12.2l0 5.1l.200 5121JO 5.121110 6.12.200 6.12.200 612.200 Ttme 1140 hri t350 hri 1445 hri 16l0 M 1a.0 hrs 1200 hri t430hrs 151511$ 1645 hrs 0745 hrs 6 6 6 6 6 6 6 8 8 AID!ude (ft) 10208 9931 9340 8956 1728 9960 9370 9000 9015 8!85 rm~ 0900 hrs @l5 hts 1100 lv$ 1215 hts 1410 hrs 1545 hts 163llvs 0000 hrs 0850 hrs 1030 hrs l.!tittJd! 27'4Bil 27'-IB'3l 27'4558 27'-1~42 27'-18'83 27'-18'25 27'45'70 27'45!0 27'45'37 27'-15'41 Altitude 1ft) 8948 9195 9017 8814 764; am BB5l 11150 am 8937 9'N 6'N 4'N O'N 9'N 4'11 7'N 1'N 4'N 4'N l.alillde 27'45'272 27'4497 21'1<4'40 21'-13~ 27'42'75 27'4283 27'43'38 27'4400 27'4~48 27'4~54 Lcngitude B8'317ll 811'3165 IIB'371J1 118'32'33 68'3779 811'31'01 811'31'72 88'3117 811'32'50 8!"3216 'N 7'N 7'N 1'N B'N ~N S'N 2'N 5'N 4'N O'E 9'E ~E 9'E O'E t'E 9'E re re r= L.ongitud• sao-.a·54o lll'3258 ssomo 911'33m 91l'lr83 911'33~7 9~11 ssom1 ~49 8803292 Soil c~our SliWI1 BroNil SliWI1 Dark BrtlWl Brown Oa!k Dark Brown Brown 'E 4'E 2'E ~E 9'E 9'E 5'E ~ 5'E l'E brotm btOII!l brown Soil colrur Brown Brown Brown Dark Brown Dark Black Brown B"""' B"""' :IJ Soil temp 380 100 tO» 5.0 tO.O t80 6» 5» 5.0 20 brown brown m (/) ('C) Soil temp ('C) 4.0 60 4.0 10.0 9.0 96 45 62 4.5 45 c Soil 40 42 25 10 ro 20 15 16 15 90 Soil moisture 18 3J 65 30 55 10 35 ill 20 60 !::; rmistiJe (%) (/) (%) )> Soil pH 6~ z Soil pH 68 6.8 6.6 6.6 6.6 6.7 6.6 45 615 6.4 6.4 66 68 6.5 69 6.7 63 6.8 64 0 Air Air ~mp('C) 6.0 1lD B.O 13.0 10.Q 10.0 6.0 60 4.0 11.0 0 temp 15 17 6 4 IB 24 1 6 4 2 1ii ('C) Humidly(%) 30 24 20 11 22 27 30 31 33 3; () c Humidlf 5 5 5 5B 7 5 12 12 22 30 Asp•ct East ~ Sllllh South Noril North Sruh South Noril tlJrth (/) (II) East East West East East East east East (/) B Asped SE SE se SE SE SE &N SE se SE TopJgraphy fi!ly fllo/ fi!ly Wen Hily Hlo/ road Hit~ river "'"' Plain Hlly z Topog~ptr,o vm fi!ly l!1ti Hil~l riwr HiDy Hit¥ Hi~ rivet Hi}( Hill Hi~ reardty draineJ sije side vale)' h~ side side !Men! ~~ Ste t!Mr 21-40 21-40 4180 21-40 21-40 21-40 61-80 20 20 21-40 Srre CMr 21-40 61-111 21-40 21-4] 21-40 21-4) 21-40 41-lll 21-40 21-40 (II) (%)ja0 plantsj Vegetation Foresl Forest Forest Foresl Fomt For!st Vlbge G~ting Forest WeD Veg!taticn l)tle ForEst ForEst ForEst Forest Foresl Forest Forest 0~ Forest l)tle roadside Fmst tlr!ined side Forest forest "'"'ftrest Manageme11 Natur.l Nalnl Nalurll Nallral Natural Natural Nalurll N

Hippophae salicifolia D. 189 94.50 100.0 31.51 72.07 45.9 24.4 142.5 Don 9 Daphni cannabiana Wall. 37 18.50 40.00 1.20 2.74 9.00 9.76 21.50 Alnus nepalensis D. Don 30 15.00 15.00 1.37 3.14 7.30 3.66 14.10 Rhododendron sp. 28 14.00 30.00 0.86 1.98 6.81 7.32 16.11 Tsuga dumosa (D. Don) 26 13.00 40.00 5.07 11.59 6.33 9.76 27.67 Eichler Populus deltoids Bartr. 24 12.00 25.00 1.10 2.51 5.84 6.10 14.45 Clzenga sp. 13 6.50 10.00 0.26 0.58 3.16 2.44 6.19 Leucosceptrnm cannum 13 6.50 15.00 0.26 0.58 3.16 3.66 7.41 Smith Betula uti/is D. Don 9 4.50 25.00 0.33 0.76 2.19 6.10 9.05 Lonicera glabrata Wall. 8 4.00 10.00 0.31 0.72 1.95 2.44 5.10 Taxus baccata L. 8 4.00 15.00 0.47 1.08 1.95 3.66 6.68 Pteris villosa Windham 7 3.50 20.00 0.14 0.31 1.70 4.88 6.90 Rubus kumaonensis 7 3.50 15.00 0.29 0.67 1.70 3.66 6.04 Balakrishnan Auracaria sp. 3 1.50 15.00 0.22 0.49 0.73 3.66 4.88 Elsholtzia jlava 2 1.00 5.00 0.04 0.09 0.49 1.22 1.80 (Benth.) Benth. Acer cappadocicum Gled. 2 1.00 5.00 0.04 0.09 0.49 1.22 1.80 Elaeagnus Elaeagnus L. 0.50 5.00 0.02 0.04 0.24 1.22 1.51 Juniperus recurva Buch.­ 0.50 5.00 0.02 0.04 0.24 1.22 1.5 I Ham. ex D. Don Populus gamblei Dode 0.50 5.00 0.18 0.40 0.24 1.22 1.87 Piptanthus nepa/ansis 0.50 5.00 0.02 0.04 0.24 1.22 1.51 (Hook.) D. Don Betula alnoides D. Don 0.50 5.00 0.02 0.04 0.24 1.22 1.51 (wide distribution of species among the to assess floral diversity and species same plots) and Importance Value association with Hippophae salicifolia Index or IVI, which is the expression in all the quadrates referred above and of reasonable measure to assess the expressed at fig. 4.3. IVI also reflected overall significance of species by the overall importance of each species taking different properties of species in in the community structure of the study vegetation (Phillips, 1959), were the area. important attributes considered along In table 4.6 we can see common plant with other parameters like Abundance associates of H. salicifolia and also the (A), Basal area of each tree species per pattern of change in IVI of twenty one hectare (BA), Relative density (RD) tree species of the study site. Of all the and relative frequency (RF). IVI tree species Hippophae salicifolia among all was the important parameter recorded the highest IVI value (142.45) RESULTS AND DISCUSSION 81 160

m p 140 0 ,~ r •n 100 c

I ·-v VI a~ =! u 40 e 20 n 0 I I. • I I d Lilli • • •

Table ~.7 : Results of ~oil anal} sis

l"t\1\"ERSIT\' OF t\ORTH BE~CAL ln,titute of Plantation rience & lanagement (Tea ~ l anagl' lll e nt ) . P.O: onh Bengal Uni,er, it) Raja Rammohunpur. iliguri - 1J~ O IJ D i~ t. : Darjeeling. \\'est Benga l. India Phone: (!J: )- ~699-t 1 5 ((}) (Tea Board, Cow rnment of India, ApproH•d Soil T{' ling Laborator)·) OIL TESTI~C REPORT

Reference o: PGDHI/ 30 / 0 /377 Name of the Garden/ Pany: Mr B. C. Basi,tha Date: 12.W.2008 /.ubi .'lam pit t:lrctricnl lk,:a11r ()f!:Q/11( S11ro,;t 11 tl rmlablt A 1'111/ahlt th'DIIablt C/J~idt .'wit rlo.r 'wind rodt Collllrtcth·ity roriHm ma/trr '} I. ! J PfJ, mlpl111rp mr:IJ: r.· '• 'f IILmlwfon '< r;. ppm PJIIII pm mn- Q l 0~6 2. 19 178 OJO .IX.t\ 2176 11.78 0.00! ~ .00 3.100 6!00 16!96 Q2 0. 16 0.57 0.9 0.08 16. !5 !6. 11 !!.59 0.005 3.00 28.00 6900 QJ 0.!3 !.OS 3.59 0.!9 J6.91 21.68 2Jj7 O.OOJ 5.00 :10.00 65.00 QJ 0 II 0.78 13.1 0 11 1105.1 I ~ ..!J !1.50 0.007 ! .00 !0.00 7!1.00 Q5 0!6 0.91 1.67 on 1S.OS !9J8 25J5 0.006 2.00 !7.00 71.00 Q6 0 19 0.J9 0.8J 0.07 8J 79 JJ5! !651 0.005 JOO 26.00 7000 Q7 0 IJ 0.6-1 1 10 009 9.\9J !667 2553 OW! 100 2100 75.00 Q 0 ! 1.0.\ 17 OIJ J! 70 65.!9 !J I! 0.005 2.00 .\• . 00 t>.\m Q 9 O.IJ I. OS 1.86 0. 15 t.J 05 27.! 1 29J6 O.OOJ 200 !6.00 7!00 QIO OJI 0.99 1.7 1 O.IJ 67.10 26. 17 33.38 0.007 ~ .00 JO.OO b6.00 Qll 0.{)(. 0.86 I J 0. 1! 71 98 1..17 39~ O.W! JOO \.100 b! 00 Q I! 0.16 0.91 1.67 0.1.1 Sl l.l !9.66 J I.!J 0.006 2.00 \.1 00 bJOO Q 13 0.23 0.86 I J 0.12 78.08 19.58 265 1 0.005 J .OO .\ 1.00 b5.00 Q IJ O.!J 0.95 1.6-1 0.11 7!.1 .\099 .\5.35 O.OOJ 6.1KI .\.1.00 61.110 Q 15 OJ J O.J I 0.7 1 0.~ IJ6.JO 2 (>. ~8 n5o 0.00() 5.00 .\ 1.00 6JOO Q 16 0 !9 J!J 5.59 0 J5 91 53 2b!r7 21.60 0.007 ! .00 ~800 7U lXI Q 17 0 1J 0.. \5 0.60 0.05 lllQ.SO 18.09 3.1.18 0.005 J.OO 10.00 b600 Q 18 0.2.1 UJ l iJ 0.17 I I ~ 9! 28.!9 26.51 0.00'1 2.00 1(100 1>8.00 Q 19 IJ.Io! ,1. ~6 h. IJ O.J'J lW 79 2J 65 26.66 O.W! 1.00 \000 67.00 Q :!O 0.15 170 a 5 0 17 109 19 \OJ6 .\5.16 0.005 J.OO ~6 . 00 7000 RESULTS AND DISCUSSION 82 with Rdom=72.07, RD=45.99 and RF cannabiana Wall (IV1=21.50) and contributing 24.39. This species had Rhododendron sp. (lVI= 16.11) were homogeneous patches in the study site, found to be very closely associated which can be matched with the tabular with Hippophae salicifolia data showing very good presence i.e., (1¥1=142.45). Juniperus recurva Buch. 100% frequency. Out of all the tree -Ham, ex D. Don (IVI=l.51), species, Tsuga dumosa D. Don Eichler Elsholtzia flava (Benth.) Benth, (IVI=27.67), Daphniphyllum (IVI=l.80), Piptanthus nepa/ensis (Hook.) D. Don (IVI=1.51) and Betula alnoides D. Don (IVI=1.51) showed greater disturbance and much lower IVI in stands of the sample plot. Thus this gives information about the most important species m the sample systems.

4.1.6 Soil analysis:

V1 The result of soil analysis is shown at -':jj 00 table 4. 7. Soil moisture and pH can be \0 _,; referred from the table 4.5. Organic matter and nitrogen percentages ranged from 0.60-6.14% and 0.05-0.49% in Q 17 and Q 19, respectively. Soil moisture ranged from 10% at Q 3 to 90% Q18 below Chaten with gorgeous shady terrain. Statistically there was - V1 1 00 not much variation m soil pH of ~ different Quadrates m all the forest \0 - types. The percentage of organic carbon (OC) ranged from 0.35% at Q17 to 3.56% at Ql9. Similarly, the .§ percentage of silt, clay and sand ranged -~ from 2-6%, 20-35% and 61-78%, § z respectively. Available potassium, RESULTS AND DISCUSSION phosphorous and sulphur ranged from of H. salicifolia.

38.43ppm (Ql) to 146.40ppm (Q, 15), There are reports that major 15.23ppm (Q4) to 76.17ppm (Q12) and proportions of phosphorous in the soil 11.78ppm (Q1) to 41.24ppm (Ql2), are stored in the forms, which are not respectively. Chloride ranged from available to the plants (Murphy, 1958). 0.002 (Q, 1) to 0.009mg/g (Q, 18). The This phenomenon compliments the electrical conductivity (EC) which explanation of low phosphorous plays a vital role in germination of content (15.23-76.17ppm) in different seeds in the soil, ranged from 0.06 soil samples collected from the study (Qll)-0.62m.mho/cm. EC less than area. l.Om.mho/cm is regarded to be normal According to Baneljee and Chand and suitable for germination, whereas 1 (1981), low percentage of organic -2 and 2-3m.mho/cm and above are carbon in the soil is an indicative of critical for germination and critical for forest fire in the past. The amount of salt sensitive crops. Above 3m mho/em organic carbon revealed from the soil is injurious to all crops (Jackson, test report of my study area suggests 1979). that there may have been a forest fire in However, from the analysis it is the past. I have found higher amount of observed that there was not much organic carbon percentage in the leaching of nitrogen in the soil. Though riverine area (table 4.5) compared to the soil conditions of the study site non-riverine area. were stressed with fewer amounts of 4.1:7 The plant morphology: minerals available in the soil, it was Sea buckthorn (Hippophae salicifolia) adequate for other species to succeed plants are erect, bushy, shrubby tree and regenerate due to the presence of (3ft.-4ft.) or trees up to 35ft. (fig 1.2A), this actinorhizal species. Among all the with average standard deviation (SD) quadrates, Ql9, the area below Lachen with dense H. salicifolia old forest was height of 16.45±4.62 in riverine and 10.67±3.69 in the non-riverine areas. observed to be more adequate for The age of plant was estimated to be regeneration and succession of other 4.69±1.18 in riverine and 4.25±1.12 in plant species along with H. salicifolia. the non-riverine areas (table 4.8). The Overall, the soil content of the study trunk was brown in colour with its area is suitable for natural regeneration RESULTS AND DISCUSSION circumference ranging from 10 em to op1mon that morphological changes 3.5ft. at chest breast height (CBH). may be related to soil moisture and However, unlike in my study area, distribution, and thus such minor Hippophae salicifolia tree with a changes might have also been observed height of 56ft. tall has been reported in plants located at different ecotypes from Mustang by Rajchal, (1998). of my study area. Such minute detail

The young branches that looked analysis during ecological studies reddish brown to green in some cases makes the study more interesting. and was profuse, alternate, unevenly Repeated visits to the study area m distant and sometimes whorled. different seasons revealed that during Tillering was ·seen in many trees, spnng and summer seasons, the shrubby and bushy plants. New plant numbers of thorns increased, looked growth was observed mainly in bushy greenish and were spiny. The same had plants from a rhizomatic stolon like reduced in number with their maturity connectivity resulting to a growth of a due to branching (with leaves) or new plant. This was rarely seen in breakage in the autunm and winter higher plants. The canopy size varied seasons. The reason behind increase in from 11.45±4.26 to 10.67±3.69, number of thorns during early season irrespective of height and development may be due to the emergence of young of the plant. flushes and for protection of the

From the table 4.9a and table 4.9b, it younger fruits before they were was interesting to observe that average matured. It was also observed that the leaf length (up to 14.4cm) and average trees species were less thorny than the inter-nodular distance (up to 14.7cm) shrubby or bushy species. In some area were more in plants growing in non­ of Zema II and its adjoining river bank, riverine areas compared to riverine Hippophae salicifolia had sought plants. Similarly, average leaf width branches and a big foliage canopy. In (up to 2.35 em), average pedicel length Lachen town, near the monastery male (up to 0.48 em) and average inter­ and female trees measuring 35ft. and branch distance (up to 82.80 inches), 25ft. with trunk diameter of 4.2ft. and were observed to be more in plants 3.5ft., respectively, has been standing growing in and around riverine areas. (fig. 1.2A). Holland and Steyn (1975) are of the RESULTS AND DISCUSSION ss

Table 4.9a: Biophysical parameters of randomly selected Hippophae sa/icifolia from riverine areas of the study area Plant location Average Average Average Average Inter Average inter- leaf length leaf width pedicel Branch dis· nodular dis- (em) (em) length (em) tance (Inch) lance (em) Riverine 9.43 0.97 0.36 29.85 6.7 Riverine 5.75 1.27 0.38 27.43 8.2 Riverine 6.47 1.41 0.34 24.26 3.9 Riverine 9.41 2.35 0.47 22.40 4.5 Riverine 8.50 1.41 0.36 20.32 4.5 Riverine 10.30 1.63 0.45 28.15 3.2 Riverine 5.80 1.42 0.42 82.80 6.5 Riverine 8.67 1.40 0.39 12.90 13.2 Riverine 10.11 1.45 0.41 22.00 4.1 Riverine 7.00 1.10 0.29 14.00 2.3 Riverine 9.33 1.22 0.25 28.89 3.4 Riverine 9.23. 1.34. 0.32 16.43 4.5 Riverine 8.75 1.79 0.33 27.35 5.7 Riverine 10.25 1.87 0.38 24.25 5.2 Riverine 8.76 1.29 0.42 12.80 4.3 Riverine 9.15 0.91 0.35 21.45 3.4 Riverine 8.97 1.37 0.48 25.66 3.2 Riverine 9.88 1.42 0.34 31.02 4.2 Riverine 8.79 1.56 0.37 26.64 3.8 Riverine 7.54 1.45 0.33 23.79 3.6 Table 4.9b: Biophysical parameters from non-riverine areas of the study area Plant location Average Average Average Average inter Average inter leaf length leaf width pedicel branch dis- nodular dis- (em) (em) length (em) tance (Inch) tance (em)

Non Riverine 7.52 1.36 0.43 24.26 14.7 Non Riverine 7.25 1.38 0.46 13.10 5.6 Non Riverine 14.41 1.16 0.44 20.68 5.4 Non Riverine 7.33 1.00 0.27 35.24 3.2 Non Riverine 8.76 1.13 0.41 36.49 2.9 Non Riverine 8.02 1.37 0.39 16.71 2.8 Non Riverine 9.53 1.32 0.39 13.72 11.3 Non Riverine 6.41 1.56 0.33 26.04 5.6 Non Riverine 6.37 1.36 0.40 17.72 6.0 Non Riverine 7.36 1.46 0.38 18.75 3.6 Non Riverine 12.45 1.35 0.42 17.41 11.2 Non Riverine 7.37 1.32 0.38 26.11 7.9 Non Riverine 8.12 1.17 0.43 18.56 8.5. Non Riverine 10.12 1.25 0.41 32.13 5.2 Non Riverine 8.97 1.31 0.29 12.21 4.3 Non Riverine 9.45 1.28 0.38 34.26 2.8 Non Riverine 8.96 1.26 0.42 18.23. 4.4 Non Riverine 8.65 1.37 0.39 19.86 3.9 Non Riverine 6.74 1.31 0.34 18.26 6.1 Non Riverine 7.66 1.27 0.36 25.32 3.3 4.1.7.1 Leaf: lanceolate, acute, alternate, dorsiventral

Leaves of Hippophae salicifolia were with ventral side covered with silvery small pedicillate, entire, linear, stellate scales and pubescent hairs RESULTS AND DISCUSSION 86 giving willow like shiny feature. These current season growth and in the older stellate scales help m reducing branches. Both male and female moisture (Rongsen, 1992). Leaf size flowers had no petals and possess four varied in different growing conditions stamens in male flowers and one pistil and even in a single plant. Leaves of with conspicuous single ovary in plant growing in and around riverine female flowers. Often, floral buds were areas were bigger in size than plants found mixed with the vegetative buds growing in slightly stressed conditions, and were rarely in pure. The male which may be due to better moisture floral buds appeared slightly bigger conditions. Mean leaf length calculated than the female floral buds, which was from 100 leaves per individual plant of similar to the report of Li. and Me 20 different plants each, from riverine Loughlin (1997). and non-riverine locations revealed that With the passage of time, repeated leaf measured 5.75 to 14.4lcm in plant tagging and observation of length and 0.97 to 2.35cm in breadth. morphological characters, growth Yang et al (1999) have carried out pattern, with respect to seasons was study in the size of leaves in higher and done for three consecutive years. From lower altitudes, which states that leaf this, it was found that the species was a size of higher altitudes were bigger in regular fruit bearing plant with no size compared to lower altitudes. cases of alternate year fruit bearing Average pedicel length ranged from character, but there was plant-to-plant 0.25 em to 0.48 em (table 4.9a and and annual variations in total fruit table 4.9b). In the report of Rajchal production. The species was a distinct (1998) on Hippophae salicifolia of dioecious with male and female Mustang district of Nepal, leaves were flowers in different plants. The male to usually 3-8 em long and 0.4 em wide. female plant ratio in the natural habitat 4.1.7.2 Flower of Lachen was observed to be about

The male and female flowers were 1:20. small in size and markedly different in 4.1.7.3 Fruits and seeds

their appearance. Male floral buds Sea buckthorn fruits are unique to other consisted of 4 to 6 flowers in cluster, fruits or berries. The juicy calyx tube while the female flowers possess only or the pulp and the seeds are of one flower and rarely two in the economic importance. It was observed RESULTS AND DISCUSSION

that the fruits took 11-14 months to 2001). ripe from flowering. The ripe fruits 4.1.7.4 Root: were orange or reddish yellow in For the plants having height of 3ft.- colour and were measured 4 to 6mm in 6.5ft., roots were seen mostly confined length and 3 to 6mrn in width with test around 3ft. to5ft. in the soil. In non­ weight of 0.97 gm. A small black riverine areas, a long taproot was found pustule was observed in mature fruit, in most cases penetrating down the with a notch. Yadav et al. (2006) have surface up to 1.5ft. to 3.5ft. reported that fruit of H. salicifolia of Approximately two years old plant was Garhwal Hills, India were ovate shaped measured with root length of 3.2ft. at with its length ranging from 5.78 mm Zema III at an altitude of 10206ft. to 7.92 mrn and width from 5.51 to Roots were found restricted to 7.24 mrn in all the locations. This comparatively less depth in the soil indicates variation of the species in with maximum percentage of hnmus different ecological conditions, because and black soil compared to sandy and geographically, Sikkim falls east to the rough soils. Both main root and tap Garhwal Hills. roots were observed in the plant The fruit of Hippophae salicifolia bore species. a dark brown, shiny, globose, single A case of aerial nodulation was also seeds measuring 3.5-5 mm in length observed in Hippophae salicifolia and 2-4 mrn in breadth with 1-2.5 mrn (unpublished). Similar type of aerial thickness. The seeds look shiny due to nodulation too has been reported in the content oil in it (Fig 1.3E). The Casuarina equisitifolia by Prin and weight of 100 seeds of this plant makes Rougier (1987). In general, the root 0.970 gm. (Basistha and Adhikari, nodules (Fig 1.4) varied in size from 1 2003). em to 10 em in diameter. The root Hippophae salicifolia fruits were nodules were milky white during the persistent in some of the branches for monsoon and late monsoon but many months, even till next season observations showed that with gradual fruit matured. Viviparous germination lowering of temperature followed by from some of these shrinked, dark dry season, the colour of nodules yellow-brown fruits has also been changed to yellowish to brown due to observed and reported (Basistha, secretion of phenolic compounds. AESUL TS AND DISCUSSION 88

2SO ,------

200 t------~------

l SO t------~------

100 +------• Age oh the plant (years) • Height (ft )

• Ca1opy (ft .) so t------• Tdlering

• Soil pH • No. of NodJies per plant Rivenne Non-riverine

Figure 4.4: Standard Deviation (SO) graph showing quantification of nodules of H. salicifolia in relation with various parameters from ri verine and non-riverine areas of

4.1.8 Root nodule quantification nodules per individual plant and total weight of nodules per plant in grams, Study on quantitati ve anal ysis of for forest stands in riverine and non­ nodulation from 40 different individual riverine areas.The plants formed a H. salicifolia plants (20 plants each dense scrub reaching heights of up to fro m riverine and non-ri verine areas), In ri verine and selected randomly were carried out 16.45±3.85ft. l4.65±4.57ft. in the non-riverine areas. taking biophysical parameters to see if The age of the plants appeared to be any of these parameters affected 4.69±1.1 8 year in riveri ne areas and nodulation of the plant. The species 4.25± 1.12 years old in non-riverine growing in high biomass possessed areas. Root nodules were located in the lesser number of nodules compared to soil at depths ranging from 3 ern to 120 plants in the stressed conditions with ern, which were mainly located in the less biomass but the nodulation tap roots and lateral roots. Numbers of percentage was l 00%. nodules per plant in stands of H. Table 4.8 illustrates the standard salicifolia were counted as 56.0±21.93 deviation (SD) of age of H. salicifolia in riverine area and 22.2±9.5 1 in non­ trees in years, their height in feet, riverine areas and total nodule weight canopy in feet, soil pH, number of per plant was recorded as RESULTS AND DISCUSSION 89 Table 4.10: Germination performance of Hippophae salicifolia seeds in different

Soil type Days to initial Days to 50% Total germin.(%) Growth/month Germination germination (Avg.) em Black soil 18 26 40 1.9

Brown soil 17 21 80-85 0.7

Perlite 17 25 85-90 1.9

Sandy soil 13 22 80-85 1.8

125.7±102.76 gm. m riverine and Despite of ecological significance of 33.45±19.65 gm. in non-riverine areas. Hippophae L in temperate areas, there pH in this experimental finding was are very less reports on nodulation recorded between 6.63±0.21 in riverine works from the fields. In the report on and 6.68±0.15 in non-riverine areas. H. rhamnoides L. growing in coastal

Figure 4.4 and table 4.8 explain areas of Lincolnshire (England) by number and weight of nodules per Stewart and Pearson (1967) it is also plant were more in taller and older suggested that number as well as Hippophae salicifolia plants growing weight of nodules increased with age in riverine areas compared to non­ of plants up to 13 years. Oremus riverine areas. It was interesting to note (1979) has also illustrated that nodules were quantified more in Akkermans's report stating 1-2 years riverine areas than the non-riverine plants growing along the Dutch coast areas. The other factors like canopy were rich! y nodulated compared to size, tillering, pH did not show any older plants. In the course of this study, remarkable effect on nodular quantity. it was also observed that young roots In this quantification experiment, being of younger plants growing in riverine the first initiative of its kind for areas did not have necrotic nodules and Hippophae salicifolia of Sikkim, both also had more number of nodules living and necrotic nodules were compared to roots of older plants and considered with an objective to see the older roots of younger plants quantity of nodules per plant. The irrespective of their height. graph also shows the age of plant with Quantification of nodules of a remarkable standard deviation Hippophae salicifolia in the riverine compared to other parameters in the areas of Lachen valley and the results reported by Stewart and Pearson graph. (1967) and Oremus (1979) have shown RESULTS AND DISCUSSION 90

Table 4.11: Effect of GA3 on root and shoot growth of sea buckthorn seedlings Treatment (ppm) Root growth (em) Shoot growth (em) Control 2.02 2.0 600 2.80 5.75 700 6.30 8.07 750 4.85 5.00 800 4.25 4.47 CD atP 0.05 8.67 4.95 CD atP 0.01 11.9 Non-significant some similarities irrespective of conditions and also nodulate more in different parameters taken for each these conditions. I did not go through experiment. any such report on comparison of

The reason behind higher number of nodular richness in Hippophae L or nodulation and thus increase in weight other actinorhizal plants growing in of nodules per plant in the riverine riverine and non-riverine areas, but areas may be due to more moisture reports on leguminous plants suggested contents in sandy soils of river banks. that flash floods characterizes loss of It was seen during the monsoon that nitrogen from the soil by leaching and constant rain and flash floods washed de-nitrification which is not common in plants growing in waterlogged or away the biomass of riverine areas ' thereby leaving the soil with little non-riverine areas (Barrios and Herrera nutrients compared to the soils of non­ 1994, Moreira and Franco 1994, Saur, riverine areas, which did not face et al. 1998). Nitrogen being same in all constant flash floods and had less soil cases, may have been leaching in erosions. Such natural activities made riverine sandy soils of Lachen valley the soil stressed left with little nutrition also, thereby forcing H. salicifolia to for the plant compelling it to nodulate nodulate more compared to non­ more. Reports by Schwencke and riverine plants. Further moisture too Carua (2001); Dawson (1990); Sen played a great role in gaining the (1996) also suggests that actinorhizal weight of the nodules in riverine areas. plants can grow well in stressed soil 4.2.0 GROWTH OF HOST PLANTS

Table 4.12: Response of chemicals in germination of sea buckthorn seeds

Treatment(s) Days for initial germination Days to 50% germination H,so. (I.5N) II I6 GA3 (700ppm) 08 10 IBA (!ppm) 09 11 NAA(5ppm) 09 II Control (H20) 17 20 RESULTS AN D DISCUSSION 91

I• initial • 50% o total p 90 e 80 r 70 f 60 0 50 r 40 m a 30 n 20 c 10 e 0 Sandy

Soil type

Figure 4.5: Germination of Hippoplrae sa/icifolia seeds in different soil ba cd media 4.2.1 Seed germination germination in sandy soil, which took

Propagation of seeds of Hippophae onl y 13 days. This may be due to better salicifolia wa studied in different oil oil aeration and temperature combinations. Table 4.10 presents the absorption by and compared to the other two soil tried. Better 50% details on germination and growth pattern of seedling in different soil germination of the seeds was seen in brown oil , which just took 21 days ba ed media in the field during the month of May-June at Gangtok compared to other soil combination . (4,700ft.) with temperature ranging Germination of 85-90% was seen in perlite soil with better average growth from (1992) ( 1.9 em per month), but perlite being reported that only 13.2% ea buckthorn an arti ficial media may not be cost seeds germinate at 12°C over a period of 47 days but if the temperature is effective and readily available every between 24 to 26°C, 95% of the seeds time for rural purpose. The table 4.10 and fig. 4.5 suggest brown oil to be germinate within ix day . better fo r germination (80-85%) and Result of germination performance growth of Hippophae salicifolia eeds from the table 4. 10 shows that in the present condition. Sandy soils germination in artificial media (Kel­ may be better for initial growth but perlite) and black soil took 17 and 18 may not fulfi ll the nutritional days, respectively compared to faster RESULTS AND DISCUSSION 92

Table 4.13: Response of growth hormones in uccessful formation of callus and rooting after 90 days of plantation in sea buckthorn cuttings

Treatments (~~m ) Total rooting Total callus formation Root length (em) (Nos.) (%) (Nos.) (%) Total Mean ffi A 50 7 35 8 40 43 4.8 IBA 55 8 40 9 45 14.6 1.8 IB A 60 9 45 9 45 3 1.5 3.93

NAA 300 3 15 7 35 7.5 2.5 NAA 350 0 04 20 NAA 400 0 0 4 20

lAA SO 2 10 10 50 2.2 1. 1 IAAI OO 5 25 12 60 4.7 9.4 lAA 200 3 15 9 45 25.5 8.5

Figure 4.6: Different stages of growth of Hippophae salicifolia raised from treated seeds requ irement of the seedli ngs for later responsive for seed germination during growth and development. Black soil, the initial stage produced 50% of perlite and the sandy soils gave better germination faster than perlite, black result during the initial germination and sandy soil. Hence, table and figure period but brown soil that may not be stated above suggests that seeds RESULTS AND DISCUSSION 93 germination performance JS better in same. The seeds treated with NAA brown soil. (5ppm), rnA (lppm), H2S04 (1.5N) All the seeds after attaining the growth germinated in13, 11 and 16 days, of 2-3 inches were transferred to respectively. The roots developed after normal local soil mixed with some treatment method also successfully organic manure, showed good growth produced nodules in a similar manner performance in the hardening shed (fig. to those at natural habitat in their later 4.6). stages, when they were transplanted to sandy soil-based media inlO inches 4.2.2 Germination performance using poly-bag. active substances: The findings at table 4.12 also states Study of germination performance was that when H. salicifolia seed were carried out in two different ways as a subjected to different physiologically result two types of data have been active substances to break dormancy, obtained as an outcome of these two the response of GA3 (700ppm) was experiments on seed germination and positive in reducing the time span of growth of seedlings. The results are germination of seeds, which took only illustrated in table 4.11 and table 4.12. 8 days compared to more days by other The findings at table 4.11 showed that treatments like H2S04 1.5N, rnA there was increased growth of roots lppm, NAA 5ppm and control (H20). and shoots of the sea buckthorn seeds Subedi and Adhikari (200 1) reported when treated with at 700ppm GA3, that soaking seeds for 48 hrs. followed compared to other concentrations (600, by hot water treatment gave better 750 and 800ppm GA3).The root growth germination results. Koller et al. was found significant both at 0.05 and (1962) reported that seedling grows 0.01 critical difference (CD), while best at that temperature where the seed shoot growth was found significant germinates, growth of roots and shoots only at 0.05 CD. significant! y increased when seeds The results in table 4.12 suggests that were soaked in plain water for 24 hrs. seeds treated with GA3 (700ppm) at 22-24°C (room temperature) and attended 50% germination by 10 days then treated with GA3 at 700ppm. In from the date of sowing while seeds in this case, seeds gave better control took 20 days' time for the performance when soaked in different RESULTS AND DISCUSSION 94 concentration of chemicals stated 4.2.3 Propagation by cuttings: above, just for 12 hrs. at room Different parameters considered for the temperature (20-25"C). results of this experiment (fig. 4.7) are It is understood from the present study as under: that the difficulty in growing plantlets • Time of plantation=August- from seeds may be reduced using September. growth regulators like GA3 and rnA at • Average length of cuttings=25 em . a specified concentration. This method Average weight of cutting=75gm. proved better germination and growth • than the naturally regenerating • Average buds per cutting=20 nos. seeds.GA3 is well known germination • Average number of damaged buds stimulator (Vijaya et al., 1996; Soyler per cuttings=10. and Khawar, 2007). As a result of GA3 • Average number of nodes on the treatment, there is a mobilization of cutting of 50cm=40. stored reserves inside seeds (Soyler Cutting position while and Khawar, 2007) and it also • planting=vertical. promotes fast disappearance of large quantity of abscisic acid (ABA)­ • Crop geometry=2 x 2m. regulated polypeptides, in dormant • Spacing (row to row) on seeds (Nicolas et al., 1997). This planting=200cm. experiment may be useful for faster • Cutting for replacement of damage/ plant regeneration related to eco- dead cuttings=15%. conservation works. Hippophae • Type of set/cutting to be used for salicifolia can be planted in other plantation=Hard wood. similar areas of altitude and habitat in the dry temperate region to gain its • Number of cuttings used=2500 multifarious economic values. Nursery • No. of cuttings required for raised seedlings of this species, through replacement=20% of 2500=500. pretreated seeds are advantageous and • Total cuttings cost effective for mass regeneration, required=2500+ 500=3000. plantation and greenery development From the results recorded after 30 days programmes at temperate mountain (one month) of cuttings treated with deserts. RESULTS AND DISCUSSION 95

Figure 4.7: Cuttings raised in different soil compo iti on after trea t m~ nts . D represents the gro wth of Hippophae salicifolia in polybag in the hardening shed aft er treated wllh IBA 60ppm different concentrations of IBA, NAA, cutti ngs.

IAA and control (H20 ), it was revealed Among all the three concentration of that cuttings treated with IBA 60ppm IBA (50ppm, 55ppm and 60ppm) u ed showed 45% total rooting and 45% of for treatment, the cuttings responded total callu formation with a signi ficant well to IBA 60ppm and responded least root length of 31.5% and showed better to IBA 50ppm. Similarly for the re ult among all the treatments u ed cuttings treated with different fo r propagation of cutting . The callu concentrations of IAA (50ppm, formation (60%) and mean root length I OOppm and 200ppm), IAA l OOppm (9.4cm) was however, ob erved to be howed better performance compared higher in IAA at 1OO ppm treated to IAA 50ppm and IAA 200ppm, RESULTS AND DISCUSSION which gave mixed results (table 4. 13). also reduces the fruiting duration of the NAA 300ppm showed 35% success in plants compared to the plants raised callus formation and 15% success in from seeds. Apart from this technique root formation with mean root length of propagation, it shall also give the of 2.5cm was among the least preferred true to type of the cultivar for concentration for the treatment of economic usages, if propagated after cuttings. Optical observation during careful selection of accession with this experiment also revealed that desired characters. though both hard wood and soft wood 4.2.4 Layering equally gave response to the growth The observations of layering showed regulators, but the survival rate of hard that 13 out of total 20 Iayerages (65%) wood was observed to be more to that showed successful rooting and foliage of soft wood. growth. It was also observed that some The findings at table 4.13 are related of the rooted branches were already with the findings of Donald (1987) nodulated with 2-6 lobed nodules, who reported that treatment of cuttings somewhat whitish in colour and intact of Pinus ellitti with 1% IBA was with the root mass. This experiment successful for root initiation. Similar was done in the field during the month type of results was also reported by of May. The layerages were collected Tsar-kova-TF (1988) and Rongsen by cutting from the mother plant in the ( 1992) at 50ppm IBA in the cuttings of field during the month of September, Hippophae rhamnoides L. and transported to the trial plot at My result provides considerable Gangtok (4,700ft. amsl) for guidance towards successful propagation experiments. Plantation propagation protocol for large scale was done in sandy soil bed of 1ft. cultivation of this species for orchard height at 0.5 x 0.5ft. plant spacing. The development under horticultural model, layerages had good success rate in the afforestation, rehabilitation and experimental plot without any conservation of degraded lands in the treatments. Similar layering high altitudes of Sikkim. This experiments have been reported by technique is also inexpensive, easier to Papp (1982). perform and less labor intensive than Agro-techniques study on H. salicifolia micro-propagation (Soni, 201 0), which revealed some interesting facts. In the RESULTS AND DISCUSSION 97 trial plot it was interesting to note that 4.3.0 ANTIOXIDANT: the seedlings of even smaller size 4.3.1 Plant Yield produced sufficient root mass and The yield of the different parts of plant formation of nodules were even high as and different extracts has been shown compared to the plant from natural in table 4.14. This table shows the habitat. Nodulation was even better in percentage of yield from male leaf, the sandy soil with sufficient gravel male bark, female leaf and female bark and organic matters added with cow extracted in water 0N), Acetone (A) dung manure. The average number of and Methanol (M). nodules from 100 seedlings (5-15cm) was recorded at 12 per plant. The root 4.3.2 Total phenolic and flavonoid mass was observed to be poor in case content of water logged soil. Phenolic compounds can equally

The present study on agro-techniques contribute to the antioxidative action. also revealed that the plant can be The amount of total phenol and flavonoids in leaf and bark of male and artificially explored to several other female of H. salicifolia, separately, are dry temperate areas of Sikkim with similar climatic conditions. Cultivation explained in table 4.15. The flavonoid of this economic and eco-friendly plant content was measured as Quercetin equivalents (QE) and phenol with scientific approaches may fulfill the requirements of high altitude concentrations were measured against Gallic acid equivalent (GAE). These inhabitants. Germination of seeds using extracts had phenolic levels ranging perlite type soil or brown soil media, from 99 to 1459 mg/g GAE. Among all use of growth regulator treated cuttings the extracts, water had the highest for mass propagation programmes and content of total phenolic and the lowest thus development of forest vegetation content was observed in acetone along with soil binding and biomass production at difficult areas, can be extract. Total phenolic content of taken as viable package of practice for methanol was intermediate between water and acetone extracts. Various sea buckthorn cultivation in Sikkim flavonoids in these extracts ran cred and thus be addition of economy to the b from 135 to 707 mg/g of QE, which marginalized farmers of the remote also followed similar pattern as terrain. RESULTS AND DISCUSSION

Table 4.14: Plant yield v/s plant part Plant Part Yield Percentage* Water (W) Acetone (A) Methanol (M) Male leaf 8.5 4 12.5 Female leaf 10.3 2.5 11.7 Male bark 11.73 3.3 12 Female bark 9.8 1.5 10.45 *Values are in triplicates phenolic content, in terms of extract. hydrazyl(DPPH) radical scavenging Highest flavonoid content was present activity, ferric reducing/antioxidant in the aqueous extract of male bark power (FRAP) and hydrogen peroxide

(707±62 mg/g QE) followed by male (H20 2) scavenging assays. leaf (419±32 mg/g QE) in comparison 4.3.4 Free radical scavenging activity to female plant parts. Similarly, phenol (DPPH method) contents also followed the same Goyal, et al., 2010 has reported that pattern, where aqueous extract of male DPPH is a stable free radical with a leaf male leaf (1454±62 mg/g GAE), characteristic absorption at 517 nm and female leaf (1459±53 mg/g GAE), this is widely used to study the radical- male bark (1453±82 mg/g GAE) and scavenging activity of natural female bark (1377±62 mg/g GAE) antioxidants. were recorded. It was found that simplest method to 4.3.3 Antioxidant activities interpret data of DPPH was by plotting Evaluation of antioxidant activities of absorbance against substrate the different type of fractions of concentration, extending the Hippophae were carried out using three concentration range beyond the end­ methods, viz, 2,2-diphenyl-1-picryl- point to define the subsequent section

Table 4.15: Concentration of phenols and flavonoids in different extracts of Hippo- phae salicifolia Phenols (pg!g GAE)* Flavonoids (pg/g QE)*

Water Acetone Methanol Water Acetone Methanol Male Leaf 1454±62 137±42 558±11 419±52 163±32 315±32

Female 1459±53 157±62 329±32 351±22 203±52 255±12 Leaf Male Bark 1453±82 99±32 276±53 707±62 135±12 267±42 Female 1377±62 107±3 447±63 415±19 139±14 347±12 Bark RESULTS AND DISCUSSION 99

"""'*'""Acetone -+-Water ----Methanol __._BHT

200 160 120 80 40 20 200 160 1:20 so 40 :20 Concentration In 1'9 Concentration In 1'9 CA) 0.5 CB) 12 E E c Q 1 a 0.4 Q ,... o.a ....= ~ ~ 0.3 u "'~ 0.6 "' l6 0.2 S"' OA -e 0 0 ~ 0.2 ~ 0.1 q; ...: 0 0 200 160 1:20 so 40 :20 :200 1EO 120 80 40 :20 Concentration In 1'9 Concentration In 1'9 (C) CD)

Figure 4.8: Reducing power assay of (A) Male leaf, (B) Female leaf, (C) Male bark and (D) Female bark compared with standard (BHT) at 700nm. Values are in triplicates±standard deviation of the plot to enable more accuracy in showed highest scavenging in water defining the intersection point. This extract followed by female leaf (82%), allowed any residual colour from the male bark (84%) and female bark reduced DPPH and also any inherent (83%) at 0.6, 0.6, 0.4 and 0.8 mg/rnl, absorbance from the substrate itself at a respectively. At the same time, acetone working wavelength. For definiteness, extract of H. salicifolia plant parts the substrate concentration used had to could not reach 50% level. 0.8 mg/rnl be in the reaction cuvette in absence of concentration of methanol extracts any DPPH and alternatively the containing male leaf and female bark amount in moles of substrate added to also showed 90% quenching ability. the reaction vessel can be used. Male bark (1.0 mg/ml) and female leaf

Table 4.16, 4.17, 4.18, and 4.19 (1.0 mg/rnl) expressed 88% and 83%. illustrates the result of DPPH assay. It was found that these methanolic The plants showed high antioxidant extracts of H. salicifolia showed capacity, quenching ability ranged excellent activities with the from 5%-90%. Male leaf (86%) concentrations tested (Ara and Nur 2009). RESULTS AND DISCUSSION 100

4.3.5 Reducing Power Assay the formation of lipid peroxides,

Another significant indicator of thereby preventing liver injuries. In antioxidant,· i.e. the reducing capacity another report by Goyal et al. (20 10) it of the extract, was also observed at an is mentioned that the reducing capacity appreciable level. Fe3+/Fe2+ of a compound may serve as a transformation (by an electron significant indicator of its potential donation) activity was investigated in antioxidant activity. This IS an the samples for measurement of indicative of medicinal property of sea reductive ability. The amount of Fe2+ buckthorn. complex can be examined by 4.3.6 HzOz radical scavenging measuring the formation of Perl's blue activity at 700 nm (Ebrahimzadeh et al., 2010). Tables 4.16-4.19 illustrates that the Increase in absorbance was an ability of quenching H20 2 in methanol indicative of increase in reductive extract of male leaf (100% ), female activity. Figure 4.8 showed the leaf (99%), male bark (100%), female reducing power of different parts of H. bark (98%) was higher compared to salicifolia ranging from 0.021 to 1.041 acetone and water extract. Table 4.20 Abs at concentrations ranging from 20- showed the scavenging of hydrogen 200 J.!g/ml of the extract. The reducing peroxide gradually increased with the capacity of extracts in different plant increase in concentration of the extract parts of H. salicifolia were as follows: as well as the standard (BHT). water (W) > methanol (M) > acetone 4.3.7 Correlation study (A). Water extract of male bark (1.04) Observations made in the past clearly showed the highest reducing activity. indicates that there is a close In different plant parts, reducing power association between overall phenolic from highest to lowest range were as content and antioxidant activity, like follows: Male bark (W) > Female bark reducing power and radical scavenging (M)>Male leaf (M)>Male leaf(W) effect on DPPH radicals (Li et al., >Female bark(W)> Female leaf(W) 2003). Similarly, indications that >Male leaf(A)>Female bark(A) correlation coefficient can be used as >Female leaf(A)>Male bark(A). an indicator of antioxidant capacity Report by Okuda et al. (1983) states were established after linear that reducing power of tannin inhibits RESULTS AND DISCUSSION 101 relationships between these parameters positive linear correlation was had been found for L. cuneata extract established between phenol content and (Kim and Kim, 2007). From our DPPH. (Linear correlation coefficient experimental observations it was found of Male Leaf R2 (W)=0.7273, R2 (M) that there were high correlations =0.8451, R2 (A)=0.674l, Female Bark between the phenolic levels of H. R2 (W)=0.8845, R2 (M)=0.5409, R2(A) salicifolia extract and antioxidant =0.7024, Male Bark R2(W)=0.4623, activities, which suggested that R2 (M)=0.9144, R2(A)=0.4462, Female phenolic compounds acted as Leaf R2(W)=0.8386, R2(M)=0.9256, R2 antioxidants in this plant species. (A)=0.9354 (figure 4.9).

To determine relationship between To evaluate antioxidant activity of total antioxidant activity and phenols of H. phenolics and flavonoides in this study, salicifolia extract and fractions, a three different kinds of extracts

~Acetone 'j 0.11195:.: + 31.3.!2 120 100 V"" D.2ce5x ·20.co4 = y =0.0231x + 39.M5 R"=Oll386 V =O.CE66X + 33.7 90 R'•0.92S6 R'=0.7273 ~100 ~=0.8451 "' "" • •• "' ..."c 70 • .."> 80 !,\! • ... .. 60 tn"' EO 0)" 50 :.: :.: ...a. ... 40 c All ...c 30 ~ • ~ 20 20 'I= OA908x -42.!()7 •y:::: 0,3049x -29.965 R'•0.6741 10 R'=0.9354 0 0 0 !00 1000 11<10 2000 2500 3000 0 roo 1{)00 1500 2000 2503 3000 Phenol Concentration (in ll"!J GAE equivUonl) Phenol Concentration pn mg GAE equiVIIantl (A) (B)

120 ,. 0.3071x ·~ 120 y:~0.120Sx+ 17.355 R'=0.54(J9 R'=0.9144 y = 0.0335x + 18.474 y =0.0121x-t ST,CSG 100 r., R'= 0~£45 80 + ·.~ ~ ".. • ~ • . ... 80 :.:"' • a. 40 I• ...c ~ 20 • y= 0.4032x ·26.542 t··R'=0.7024 opL-.---.---.--.---.--. 0 o roo 1000 1500 2000 2600 3000 0 500 1000 1500 lOOO 2500 Phenol concentrallon ~n mg GAE equiVIIantl Phenol Concentrallon (in mg GAE equlvllanQ (C) (0) Figure 4.9: Correlation between DPPH and total phenol content in water, methanolic and acetone extract of (A) Male leaf, (B) Female leaf, (C) Male bark and (D) Female bark. Values are in tripli­ cates±standard deviation RESULTS AND DISCUSSION 102

Table 4.16: Percentage inhibition* (%!)of radical scavenging of male leaf of Hie.e.oe.hae salici(plia Extract concen· Male Leaf !ration (mglml) DPPH HzOz Water Acetone Methanol Water Acetone Methanol 0.1 36±21 6±67 33±21 5±53 57±28 92±23 0.2 63±55 12±51 59±11 51±23 65 ±71 94±30 0.4 86±89 18±71 71±26 67±51 75±50 96 ±71 0.6 85±12 32±12 86±30 78±55 92±20 97±24 0.8 84±10 35±76 90±66 89±21 83±40 99±53 1.0 83±12 45±26 89±21 95±10 96±11 100±33 Table 4.17: Percentage inhibition* (%!)of radical scavenging of female leaf of Hip­ pophae salicifolia Extract concen· Female Leaf !ration (mglml) DPPH HzOz Water Acetone Methanol Water Acetone Methanol 0.1 23±61 4±41 12±66 54±11 80±28 93±54 0.2 43±10 8±91 23±21 64±91 85±26 95±11 0.4 72±12 16±80 45±90 73±32 89±18 96±33 0.6 82±31 22±97 59±25 82±44 92±60 97±43 0.8 79±61 27±88 72±33 90±30 96±20 98±21 1.0 82±21 30±12 83±22 95±13 98±34 99±81 Table4.18: Percentage inhibition* (%!) of radical scavenging of male bark of Hippophae salicifolia Extract concen· Male Bark !ration (mglml) DPPH HzOz Water Acetone Methanol Water Acetone Methanol 0.1 48±61 4±20 16±16 5±7 57±65 92±32 0.2 77±81 7±62 31±40 51±10 65±98 94±40 0.4 84±67 13±61 55±10 67±17 75±91 96±89 0.6 82±77 18±56 75±20 78±66 83±17 97±21 0.8 80±17 23±69 87±56 89±99 92±18 99±42 1.0 78+18 28+61 88±78 95±53 96±12 100±64 Table 4.19: Percentage inhibition* (%!) of radical scavenging of female bark of Hippophae salicifolia

Extract concen~ Female Bark !ration (mglml) DPPH HzOz Water Acetone Methanol Water Acetone Methanol 0.1 23±61 5±11 26±11 54±34 93±76 80±55 0.2 46±16 8±17 46±41 64±16 95±16 85±68 0.4 74±61 14±78 79±99 73±64 96±10 89±65 0.6 83±56 23±67 89±53 82±83 95±62 92±44 0.8 82±67 28±22 90±89 90±77 94±53 96±65 1.0 80±31 33±91 89+16 95±7 94±10 98±47 *Values are in triplicates±standard deviation (for all the tables of this page) RESULTS AND DISCUSSION 103 Table 4.20: Radical scavenging activities of BHT as standard at different concentrations Standard concentration (mglml) DPPH HzOz 0.1 33±11 34±4 0.2 46±6 43±6 0.4 58±8 57±16 0.6 73±15 62±13 0.8 82±36 70±8 1.0 90±10 75±16 *Values are in triplicates±standard deviation (aqueous, methanolic and acetone) inhibiting membrane bound enzymes were used from four different parts of like ATPase and phospholipase A2, H. salicifolia. Similar study of which may explain the antioxidative antioxidant evaluation was also carried action of H. salicifolia. Ferguson out by Negi, et al. (2005) in Hippophae (200 1) suggested that flavonoides rhamnoides. It is for the first time that contain anion radicals and hence serves this experiment has contributed to a as health promoting compounds, which better understanding of antioxidant supports the ethno-traditional use of properties of different parts in H. this plant in treating stress related salicifolia of Sikkim. problems and skin ailments like

In my study the presence of phenolic treating bums, scabis, cracking of skin, and flavonoid compounds of all the impetigo, keratosis, cures xeroderma parts were more in aqueous extracts and prickly heat (Negi et al., 2005). compared to methanol and acetone After incubation for 20 minutes, the extracts, which means these DPPH scavenging activity of aqueous compounds are hydrophilic and thus and methanol extracts were higher seeps into water during extraction compared to acetone extracts. As the process (Ayansor, et al., 2010) or the concentration of the extract ranging phenolic and flavnoid contents were from 0.1 to 1.0 mg/ml was gradually more in aqueous extract may be due to increased, the percentage inhibition of the difference in polarity of different DPPH too gradually increased and solvents under study (Kostic et al., similar activity was also exhibited by 2010). the standard (BHT). This may be due

Li et al. (2003) reported that flavonoids to elevated levels of the active affect membrane permeability by compounds along with the polyphenols in both aqueous and the methanolic RESULTS AND DISCUSSION 104 extracts, which have higher hydrogen that there was a good linear correlation donating ability to scavenge DPPH between total phenolic compound radicals attributing their higher content and the antioxidant activity in antioxidant activity (Chueng et al., different plants. Nagai et al. (2004) 2003) reported that there was also no

Hydrogen peroxides are highly significant relationships between the diffusible and an important Reactive antioxidant activities (determined using Oxygen Species (ROS) because of its three different methods namely FRAP, ability to penetrate biological DPPH and carotene bleaching assays) membranes and most importantly it and total content of phenolic causes activation of nuclear compounds shown by some other translocation of transcription factors researcher. Therefore, this positive NF-KB, which subsequently allows the linear correlation showed that the transcription of genes and leads to the sample with highest total phenol inflammation and Syndrome X (Goyal content likewise had the highest DPPH et al., 2010; Ghaisas et al., 2008), values, whereas the sample with lowest though, it may be toxic if converted to total phenol content produces the hydroxyl radical in the cell (Waug et lowest reducing power values. Ailments such as diabetes mellitus, al., 1998). Scavenging of H20 2 by the plant extracts may be ascribed to their cancer, liver diseases, renal failure and phenolics, which donate electron to degenerative diseases are as a result of HzOz, thus reducing it to water. deficient natural antioxidant defence mechanism. Such diseases can be cured This is the first study of its kind by polyphenols, which are the principal showing male bark had high level of plant compounds that have antioxidant reducing capacity, may be because of activity (Middha et al., 2009). Table more phenolic compounds, which are 4.14 exhibits aqueous and methanolic potent antioxidanis. The outcome also fractions containing the highest showed that there was increase in amounts of phenolic compounds and reducing power of the plant extract both fractions exhibited higher along with mcrease in extract antioxidant activities. The antioxidant concentration (Akinpelu et al., 20 10). activities of phenolic compounds were Studies carried out by Kim and Kim primarily due to their redox properties, (2007); Haraguchi et al. (1992) found RESULTS AND DISCUSSION 105 Table 4.21: Quantification of DNA isolated from the root nodules of different sam­ ples after purification and the quantity taken for PCR Sample A26o Azso A"o/A"o Amount of Amount of DNA for PCR code No nm nm DNA (ng/11l) PCR (ng/pl) Result SIHF01 O.D75 0.043 1.74 375 46.87 Band visible SIHF02 0.064 0.036 1.77 320 45.71 -do- SIHF03 0.155 0.085 1.82 775 45.58 -do- SIHF04 0.093 0.054 1.72 465 46.50 -do- SIHF05 0.159 0.089 1.78 795 46.76 -do- SIHF06 0.080 0.44 1.81 400 44.44 -do- SIHF07 0.029 0.016 1.81 145 36.25 -do- S1HF08 0.044 0.025 1.76 220 40.00 -do- SIHF09 0.038 0,022 1.72 190 38.00 -do- SIHFIO 0.213 0.120 1.77 1065 46.30 -do- SIHFll 0.028 0,016 1.75 140 46.60 -do- which allowed them to function as elimination of microbial contaminants reducing agents, hydrogen donors and associated with the nodule surface. singlet-oxygen quenchers (Goyal et al., H20 2 are potential oxidizing chemical 2010). Taken together, the high agents that are efficient in eliminating correlation confirmed that the contaminants from the surface of the polyphenol constituents of H. nodule without causing harmful effect salicifolia may be responsible for its to the cortical cells where Frankia high antioxidant activity during the were confined.

DPPHassay. Frankia is a very slow growing 4.4.0 ISOLATION OF FRANK/A organism. Each Frankia colony

4.4.1 Surface sterilization originates from a single unit called positive cells and were termed as units There are different types of able to form Frankia (UFF) (Diem and microorganisms present on the surface Dommergues, 1982). Only the positive of the root nodule, which act as cells that came in contact with the contaminants and hinder the growth of nutrient media formed a colony. The Frankia in pure culture It was UFF broadly referred either to specific mandatory to remove these structures or hyphae or cluster of contaminants in order to get Frankia hyphae, which was able to grow from clusters in pure culture. Of all the the nodule. Berry and Torrey (1979) techniques applied, the technique of had already reported that Frankia serial no D at chapter ill, (3.10.2 D) originates from clusters of hyphae, gave positive result. Success of this which may be suggestive, that hypal!al technique may be due to the efficient RESULTS AND DISCUSSION 106

clusters originating from the sliced preferred carbon source compared to nodule may be more suitable for carbohydrates (Myrold, 1994). For initiating colonies than crushed nodules further sub-cultures, Qmod medium (Sen, 1996). supplemented with Lecithin also gave

4.4.2 Culture media for isolation positive results, but contaminations in this medium were of great concern. The culture medium formulation or used in isolation of new Frankia strains 4.4.3 Nodulation test is critical for success, still till date no The nodulation test conducted in H. universal or Frankia strain specific salicifolia seedlings inoculated with 45 medium has been formulated. Different days old pure culture grown in sterile kinds of media have been described by Hoagland's nitrogen free solution Lechevalier and Lechevalier (1990) showed 40-50% of the plants formed 1- and Myrold (1994) (see review section 2 sometimes 3 nodules in 30-40 days. for details). Many Frankia isolation In a similar type of study Diem et al. media are supplemented with various (1983) reported 80% nodulation in 14- chemicals and yeast extract believed to 20 days. H. salicifolia being the plant promote Frankia growth. of cold temperate region perhaps

Frankia has been found to grow well showed some hesitance to nodulate on a minimal medium containing freely in the controlled conditions of buffered salt solutions with one carbon Gangtok. It was also observed that source like succinate, pyruvate plants without root hairs hardly propionate, etc., which can be changed nodulated, which indicated and depending on media (Myrold, 1994). supported the idea that root hairs were From different types of isolation media the prerequisite for nodulation. Apart (refer Appendix I). from this it was also seen that out of 2- 3 pre nodules per plant, only 1-2 Of all the media opted for the work, developed into creamy or milky white DPM (Baker and 0 Keef, 1984) with nodules with 1-1.5mm diameter later sodium propionate as carbon source became brownish in colour. and Qmod (Lalonde and Calvert, 1979) were best suited for Frankia isolation 4.4.4 Frankia under light microscope from Hippophae salicifolia root Sections from the root nodules of H. nodules. Sodium propionate is the salicifolia were subjected to light RESULTS AND DISCUSSION 107 microscopy, which revealed that the in inducing vesicles and sporangia in section contained clusters of thin hypha most of the isolates of Alnus nepalensis in the cortical region. The nodule lobe compared to Qmod medium. In this case section had outer epidermis covering also the isolates revealed sporangia in parenchyma cells and cortical region. terminal or intercalary positions. Three The vascular bundles were surrounded months colonies produced in DPM did by a thin layer of endodermis. The not exceed more than 1mm in parenchymatous cells too had diameter. Some pinkish to off white infections along witb cortical region. pigmentation were also observed in the All tbe cortical cells showed infection colonies developed in tbe pure culture. witb dense staining. Distinct cell walls After going through the review of were visible, within which vesicles literatures by Normand and Lalonde were present in a mucilaginous (1986) and Mullin and An (1990), it material. Only the vesicles and hyphae was understood tbat still limited in tbe outer periphery could be stained information have been generated on and were viewed. The average colony Frankia compared to Rhizobium. One measured about 300f.im (fig. 4.10). reason behind tbis may be due to

Light microscopy was the preliminary limited number of isolation and means to identify Frankia based on identification of Frankia in pure morphological characters. Many culture. Since the first isolation in reports have suggested tbat Frankia are 1978, problem of obtaining pure filamentous, branched bearing vesicles culture isolates and tbeir growth (the site of nitrogenase activity) and requirements are still restricting further presence or absence of sporangia and studies on genetics, ecology and spores (Benson and Silvester, 1993; physiology of Frankia symbiosis. Even Pradhan et al., 1999). The Frankia after formulation and improvement of isolates from pure culture of H. different culture media, Frankia still salicifolia also showed mat of septate remains a slow growing organism filaments or hyphae ranging from 0.6- requiring longer period to get ready for 1mm bearing vesicles measuring experiments (Simonet et al., 1991). between 0.3-0.7mm in diameter (fig. Even after many attempts tbere are 4.11). Pradhan et al. (1999) have reports of very few isolates of H. reported that DPM was most effective salicifolia, which is yet to be described RESULTS AND DISCUSSION 108 in details. manures and composts, more roots

4.4.5 Plant infectivity test curling were observed in young and developing roots. But care had to be Infection tests of Hippophae salicifolia taken during watering where little more seedlings grown in earthen pots, watering than the optimum quantity led showed 40-50% plants formed 4-8 to damping off and death of the plants. nodules in 4-6 weeks. Similar works by When plants of 1-2 years, grown in using the actinorhizal plant as a trap controlled environment, were plants to assay plant nodulation using transferred to the experimental site pot system with solid media has been (different from its natural carried out with Alnus, Casuarina, and environment), the plants did not show Myrica (Cranell et al., 1994; Zimpfer progressive growth in the first year et al., 1997) and Ceanothus sp. (Jeong with drastic reduction on nodulation and Myrold, 2003). Dobritsa and rate to 1-2 nodules per plant. But after Novik (1992) reported that sea subsequent year there were some buckthorn has an active, systemic growth in plants as well as nodulation mechanism for feedback control of also increased to an average of 3-4 nodulation that suppresses further nodules per plant. Though survivals of nodule formation and prevents the plants were 70-80%, the nodulation excessive nodulation. Hippophae was not to the mark as it was expected. salicifolia is a plant growing naturally The environmental factor of the soil in tropical cold desert and predominant might have reduced nodulation in the in sandy soils at the river banks (Singh plant at new environment. However, it et al., 1995; Rajchal, 2009). This may IS understood that Hippophae be the probable reason for not getting salicifolia can be infected by artificial clusters of nodulation as natural habitat Frankia inoculants. in Hippophae salicifolia when grown 4.5.0 ISOLATION OF DNA OF in controlled environmental conditions. FRANK/A FROM ROOT NODULES High NaCl concentration, low phosphorous supply and high The difficulty in isolating pure culture combined N2 supply reduces of Frankia and its sluggish growth nodulation in H. rhamnoides (Jian, created major hurdles in studying the 1989). So making the soil more genetic diversity of Frankia. However, stressed by reducing the amount of with the advent of better primer RESULTS AND DISCUSSION 109 designing ability it is now possible to yields in this method was less, it develop PCR primers, which will produced whitish precipitate of nucleic amplify a section of Frankia genome acid, which after further purification from mixed cultures or from the DNA was subjected to PCR amplification. samples directly collected and isolated Bright bands could be seen on 1.5% from the environment. The amplified agarose gel with 7f!l of ethidium products are then subjected to bromide. This approach avoided the restriction digestion to get PCR-RFLP bias-inducing process of isolation of profile of wild Frankia strains. Frankia strains from field collected

As mentioned in materials and nodules. methods, DNA was isolated directly 4.5.1 Frankia DNA purification: from the root nodules collected from The root nodules contained large II different places of Lachen Valley, amounts of phenolics, RNA, proteins Sikkim. Various methods were and polysaccharides. These compounds followed but method of Bajwa et al. hinder in DNA isolation and isolated (2005) with minor modifications was crude DNA may not be suitable for found to be the most effective one. PCR amplification. Addition of CTAB Genomic DNA of Frankia can be in DNA extraction in the first instance isolated directly from the root nodules helped in elimination of large amount (Simonet et al., 1991). Extraction of of polysaccharides from DNA DNA directly from nodules and preparation. During the later stages of amplification by PCR have also been extraction, RNA could also be removed previously reported for studying by treating the sample with lOfll phylogenetic relationships between RNase (IOmg/ml) followed by equal Frankia reference strains and un­ volume of Phenol: Chloroform: /so- isolated Frankia strains in nodules of amyl alcohol (25:24:1) and Alnus, Coriaria, and Dryas species simultaneously by Chloroform: Iso­ (Rouvier et al., 1996). The method I amyl alcohol (24: I) to the samples followed was useful to examine the dissolved in 500 fll IX TE buffer, at possible genetic diversity occurring least twice. among Frankia strains of H. salicifolia 4.5.2 DNA quantification in the tropical forests of Lachen, North Frankia DNA was quantified using Sikkim. Though the quantity of DNA RESULTS AND DISCUSSION 110

Figure 4.10: Transverse section of root nodule of Hippophae salicifolia under light microscope showi ng Frankia infection. A: T. S. under I OX magnification cortical (c) and parenchymatous (p) regions are infected with Frankia; B: T. S . under 40X magnification. Infection can be seen partly in cortical cells and parenchyma region, the cell wall (c11 ") and infected regions are prominent; C: TS closer views of infected cells with I OOX magnification; vesicles are prominent inside the cell wall of each cell. 0 : it 's the closer view (le ns zoomed); prominent Frankia vesicles with hi-lobed and tri­ lobed structures are visible in the infected parenchymatous cells spectrophotometric analysis showing obtain substantial amount of pure DNA

A26o/ A280 ratio around 1.8. This ratio for PCR amplification. Table 4.21 was chosen for further PCR based illustrates the details of amount of root methods which was having less nodules taken and the total DNA impurity and gave a bright visible band isolated after purification and on subjecting the PCR product to quantification. From the table it also electrophoresis. clear that DNA to be taken for PCR

Frankia DNA extraction, purification amplification after quantification and quantification steps allowed should be in the range of 30-50ng/~.d. optimum DNA from the root nodules Though, theoretically ratio of (A26of of Hippophae salicifolia. However, A2so) 1.8 is the ideal value of ratio, in modifications made during the process my study PCR amplification was by RNase treatment al lowed me to obtained at A26ol A2so ratio values RESULTS AND DISCUSSION 111

Figure 4.11 : Microscopic view of Frankia strains i olated fro m pure culture in DPM N2 free media (Baker and 0 ' Keef, 1984 ). A & B shows the den e mat of hyphae under 40X optical lens; C & D reveals distinct septate hypha (h), terminal po rangia (ts) and intercalary sporang ia (is) with I OOX magnifi cation (oil immer io n) ranging from 1.72 to 1.82. GGGGTCCKTARGGGYY 3') was designed m our laboratory (refer 4.6.0 POLYMERASE CHAIN chapter lll) and reverse primer FGPL REACTION 2054, (5' DNA obtained from the root nodule CCGGGTITCCCCATTCGG 3') were composed of mixture of DNA (Simonet et al., 1991), was used to from the host plant, the Frankia and obtain specific amplification of pos ibly contaminating bacteria, which Frankia DNA from Hippophae may not have been eliminated during salicifolia root nodule i e lates (fig. the surface sterilization and peeling of 4.12). nodules. Hence, Frankia specifi c DNA isolated from different sets of primer were required for obtaining nodule collected from different part exclusive Frankia genome bands. of the study ite yielded -1000 bp band Forward and rever e primer ( 16 mer) on amplification with a set of forward compatible to Elaeagnus specific and rever e primers, as stated above. Frankia (5' RESULTS AND DISCUSSION 112

Distinct band in agarose gel was visible accession number and percentage of when the annealing temperature was identity obtained in BLAST set at 54 °C for 1 minute. Even 1°C programme are presented in the table mcrease or decrease m annealing 4.22. temperature effected the band Phylogenetic analysis from the figure formation. 36.25-46.76ng/f.!l 4.14 revealed that the entire 16S rRNA concentration of DNA template in 1f.ll gene sequences of H. salicifolia­ was used for PCR amplification, which Frankia isolate (inside the red box) gave distinct bands. Out of all the DNA clusters reside close other Frankia isolates subjected to PCR strains (inside the blue box) but forms amplification, 11 isolates (SIHFO 1 to a cluster of its own. Amongst the SIHFll) formed distinct bands Frankia strains, newly sequenced although few gave low signals, isolates of H. salicifolia showed close presumably because of lesser amount proximity with that of Frankia of DNA in some samples. The results EANl pee and Frankia Eullc. Both of some amplified samples in presence these strains infect the plant Elaeagnus, of DNA-molecular weight markers which belong to the family have been presented in the fig. 4.12. Elaeagnaceae. Hippophae salicifolia is 4.7.0 PHYLOGENY AND also the member of the same family. Of GENETIC DIVERSITY: the newly sequenced isolates, FR773729 formed an out group m In the present study BLAST comparison to the ten other isolates programme was used to compare the with boot strap value of 95%. The 22 sequences namely SIHFOlf to sequences used m phylogenetic SIHFllf and SIHFOlr to SIHFllr analysis their names, abbreviations, (Accession No. FR773720 to genes and GenBank accession numbers FR773741) for homology with are given in table 4.23. sequences available in the GenBank. All the sequences showed highest The phylogenetic tree in the fig 4.14 identity with 16S Ribosomal RNA also reflects a broad genetic diversity gene of an uncultured Frankia sp. among the Frankia strains of the isolate (fig. 4.13). The sequences, Elaeagnus-infective group. Reports on which were used for prepanng close phylogenetic affinity (up to 99%) phylogenetic tree, along with their among the Frankia of Hippophae and RESULTS AND DISCUSSION 113

strains have evolved from a common ancestor. Further the close proximity of the Hippophae salicifolia-Frankia can also be related and with the altitude of the study area (may refer table 4.5 table 4.22 for quadrates and accession numbers, respectively). The isolates have formed close clusters with almost similar types of altitude and climatic conditions. In a similar type of study, it Figure 4.12: Agarose gel ( 1.5%) electrophore­ sis of PCR amplified partial 16S rRNA of DNA has been revealed that altitudinal isolates from root nodules of Hippophae sa/ici­ variations affect Frankia distribution folia collected from different locations of the study area. ML=IOO bp Marker ( lx IOObp); (Jeong and Myrold, 1999; Oakley et lanes A to K represents PCR bands of SIHFO 1- SIHFII Frankia isolates al., 2004)

Elaeagnus infective group, irrespective 4.8.0 PCR-RFLP DATA ANALYSIS of their different geographical USING POPGENE SOFfWARE:

locations, have been reported by Chen From all the samples a total of 18 et al. (2008). From the fig. it is also scorable fragments were generated observed that Frankia strains of (table 4.24), out of which 11 were different host specificity (in red and polymorphic. The primer was tried blue boxes) have the tendency to form thrice and was reproducible. The close clusters with other organisms of number of polymorphic bands per close lineages. The sequences of enzyme ranged from one in Mspi to Frankia strains present in Gymnostoma five in Hpaii with an average of 2.75 and Ceanothus nodules are (say 3) polymorphic bands per phylogenetically close to Elaeagnus­ endonuclease (enzyme). These clearly infecti ve strains (Navarro et al., 1997; visible, repeatable and polymorphic Murry et al., 1997). However, in the bands were scored and used for fu rther process of diversity study of H. analysis (fig.4.15). Due to high salicifolia-Frankia strains, it can also specificity of the recognition sites, be concluded that Elaeagnus, closer cleavage pattern in agarose gel Gymnostoma, Hippophae, Ceanothus are seen when two sequences have and all Elaeagnus-specifi c Frankia higher similarity (Nei and Li, 1979) RESULTS AN D DISCUSSION 114

--·,..115-,.W.IooL·...._.. _ - --.=;·o.---~--~~~~~~------__J [ok i d* ~ ~OJ.to ry iootm•rks Jools tldp . c ~ f(~8~~--~,~~~-~~~~-n~m-h~h-~---n-u-c-co-, L~FR~n~J~7~~~------~------~

Nucleotide A4Jhabel or Life

O!!Gbr Stllnqs ~ Gtn8ank

Uncultured Frankia sp. partlai16S rRNA gene, Isolate SIHF01f GenSon• FRn J"20 I ~ ..mwa

!.OC'J! !'R7-J'7J' ~ - ... ~ bp C::~U.. !l.:"'.IU.:r [ !rV :.q-.;':.lf-20 11 C!!':N:T:O..'t ~:'.C".Jlt ·.:: e:d ~:re. -:lc.:.o !!~. ~e.::ie.:. :65 :-;.:;~ ce-e , .!3C!o:.e 5:~!0:! . :.ccrss:o:: V!iUIC..."'l i"ZY'~l\:S !lr;• .501'Jl\.C! :.;.nc-~lt:.a ed rr•:-.lua _.p. CRGA..'C~ unc-.Jlcure:d rro:::k1a op. 3aett::.• : A~tl~!'tactt:n•: A~t :. ~cb-a c: -:t: 1d4t: A-ct 1-~::r.)'C t tt.! e ~ : ~=&::Jc!.!:~ee: ; !"::-a::k":.sceee; !':er..ua: e ::v.:. :c:-~:::.e.l ' a.r;:! e!l:

:t.:::!'!.:U !.& s ! s :~. • . !.C., n:a.n:.~ .• F:ratC'.a ::,3. a :;~ Se r: , ;.. ::-7:! ~.c::. e: c".l!a: dlve:r~l~l' c t !'!.l~~~ ~ ec: o.~.5CC1.o:e!i !"::e.::~.:.o ! :J~:::j 1:1 :.o.c!';e:: ~ a :.l4 V o : S1kk"1.:, In~& ~cmt.u:. :;r:.c:...bl!• h• d at!"!ll.'lct 2 na.=c::l : t.o --o:,

.;o~"iA:. s~ :::e:s t :.!-.;;Ji-,;c :: S4!n .:... , Y..=!~=--..:1&:- ~!':. e:t :..=.s :.ae:!'.a:c:-~·, O..part.~• nt. ot ~o;.L"':.i·. U:av e !'U.:i· c t Nc:-tt. ,..,,cal. Ra; a r&CtOh:.:.np:..::. s .:.:.~ q·.:.:~ . -3::'3, : ~m :ft

/ o:o;..::. l# ~·· .:r.c Jl ~-.u:e-:1 F:a..r.ln.a ''"." /ool_t:·.~ e- ·li;'e::cr..:.c »:Q• I 1J::la-:.c•" S : Hf'):.!" / l J o1.s tlC:". s :::.;.:ce • .. ho!:t :oct :lOdiJle s " /hc3 t. •'" H.\.~;c;::h. ae 3-&l.\.Cl ! cll.a" / d.b x::e!• "taxc:l: l 81SS2" / e:lV.:.:: : :-~::.t.e l _~~l ~ / ~.g;.r,:::.- " :~~a:: a ;la::'l Va!!.e 7,·, .!:.klr;..:t.'" < ~-->,~0 / qe::e• ":E.! ::a!l'A"

/ qe::.oe: • .. .:.E3 :..:l.A"' / p!'od..:.c : • " : ES ::.lb~ scz:.ol R:U, "

- ;'C~Cqtqt.et; t.qaqat.;tt.; q.;t.:.aa q-e.e.: eq:aa. e9• 9e q.:aaee.::: :.a ':.:.:tae.qt.t<; 6: C C.Q'CQCQtC a tqgcqcoqa ct.c.&:.aqoaq act.qcCQQ'OO :c..a.ac -:. ccqa ooaagQt.O'QQ :2: qa:.c;&c~tc& a~ t.c a:.-:a t..; e--:c-::t•cq: e -::.Qq~ c:Qc- a.:-aco:o-c:a c a.a!;-;c:; q 1!: taca.a.aqqqc tgcga t a ccg c.aao;t.QQI9 cqaetcccaa aaa.qceqgtc tcaqt.t.cgga. H: t.C:V"Q"QQ't.Ct.Q eaact.c.ga cc cc;t.;- aa-. ~c; ~qa q-;.cgco;a qc-4ot~O~Q •t:..ao::.act.Q 30: CtQCQQtqa& t &cgt.t.c:c:eo ~;-eec.t;c.ae a e a ce;eec:; teae;t.eaeo •••9t.CQQ't.a 36: a : acecq&aQ ccoot.~cc:: a.ac:::t. :.;to ~q-;~c;scc:;v : : q asqoc.c;o qa ::cqqcqa:. 12: tqqq&oquq tCO't•--= ••o c;·c.a;.ceg:ac c:q;aa;ot;c ;;ct~qatc a c:tcc t. ~tct 4!- AA':Q'&;'Cttt ct CJQ Ct Qt:O'~ qc::: :.c :.gt.& ;t.qq.a.qc;q:.q tct;q:ee.at; •7eQaaq•cc ~1: •c; ~v•• t;o ctggtc;yqq -:,q;:c;.a -;.;~9 t;t.-;q&e-:;:: :-;a c':& tQ':.t. o;.;~q;c;t.q;t 'o:. c::.; q-:..:t.;-tt. Q't.ee;q-;~: ec t..; ~q-:;t. t. ct.eto;-;o t .;; ec;q;:-:o::e: q.;;tqQ.&teq 6ti: c;t. acoctvt.t c::oo c.eo:.c a~ ~·~c- ao;"t. t. ~;c cct.cc;~oc a::oa :.cc:.c;c c:.ttct.oc:o ~ 2: ;- c :cqttq;; ;ct;gtqyqa c;;c•;yt.tt.q eqq;et.;qt.t. c;te;t.t~qq

Figure 4.13: Partial 16S rRNA sequence of uncultured Frankia sp. from Hippoplzae salicifolia root nodule named as SIHFO If and its accession number provided by the GenBank

Table 4.24 al so reveals polymorphism between the range was shown by Alui ranged from 50-7 1.4%. Restriction (66.6%). The overall polymorphism of digestion enzyme Mspi and Haeiii 61 .11 % was observed. showed 50% polymorphism while Quantitative measure of genetic Hpall revealed 7 1.4% polymorphism. divergence or genetic distance An intermediate polymorphism calculated from the Nei's original RESULTS AND DISCUSSION 115

FR773720 SIHF01f SIHF04f SIHF02f

FR773724 SIH~f FR773728 SIHF09f rR773722 SIHF03f F R773725 SIHF06f FR773730 SIHF11f FR773720 SIHF07f FR773727 SIHF08f FR773729 SIHF10f CP000820 EAN1PEC CP002200 EUI1C J.408875 EUN1F II M55343 FOG CCI3 ACN CP001867 GEO 92 CP001736 KRIB 26 CP000509 NOCD CP001737 NAK 20 C P 001621 XYL CP001618 BEU AM649034 CLAVIS AJ310418 CLAVIM AP009152 KOC CP001626 MICR -- CP001738 THER AP006618 NU(.; 97 AM420293 SAC AL939106 STOCE 93 BA000030 STAV APOQ9.493 STGR AJ243871 RUB

Figure 4.14: Most parsimonious lrec (neighbour joining) showing the relationship of 16S rRNA of Hippophae salicifolia-Frankia isolates FR773720-FR773730 with other GenBank-published se­ quences. The red box indicates Hippophae salicifo/ia-Frankia isolates and the blue box indicated other Frankia isolates. Numbers at nodes indicate the bootstrap percentage cores out of 1000 repli­ cates

measure of genetic distance (Nei, genetic distance of 0.6931 between 1972) averaged 0.36 and varied from 0, HISF04 and HISFO I strains (table lowest between Frankia strains 4.25).

HISF03 & HISF02, HISF05 & The dendrogram con tructed on the HISF02, HISF05 & HISF03, HISF06 ba is of Nei' genetic di tance utilizing & HISF02, HlSF06 & HISF03 and the UPGMA method (fig. 4.16) HISF06 & HISF05 to maxtmum howed two main clusters. These RESULTS AND DISCUSSION 116 Table 4.22: Diversity analysis of the partial 16S rRNA sequences of the eleven different Hippophae salicifolia root nodule DNA samples of the present study area along with their GenBank accession numbers

Isolates Sample Length of the Maximum similarity (%) with other Accession (Location) code No sequence (nt) sequences in GenBank No. Zema Ill SIHFOI 770 96% with Uncultured Fraukia sp. iso­ FR773720 (QI*) late HIS2Hp 16S ribosomal RNA

Chaten SIHF02 687 96% with Uncultured Frankia sp. iso­ FR773721 (Ql6) late HIS2Hp 16S ribosomal RNA gene, partial (D098898 1.1 Chako SIHF03 702 96% with Uncultured Fraukia sp. iso­ FR773722 River (QI4) late HIS2Hp 16S ribosomal RNA gene, partial (D098898 I .I Dozam SIHF04 723 95% with Uncultured Fraukia sp. iso­ FR773723 (Q6) late HIS2Hp 16S ribosomal RNA gene, partial (D098898 1.1 Below La- SIHF05 732 95% with Uncultured Fraukia sp. iso­ FR773724 chen (Ql9) late HIS2Hp 16S ribosomal RNA gene, partial (D098898 1.1 Zema II B SIHF06 798 96% with Uncultured Fraukia sp. iso­ FR773725 (Q4) late HIS2Hp 16S ribosomal RNA gene, partial (D098898 1.1 Puchi (Ql3) SIHF07 539 99% with Uncultured Fraukia sp. iso­ FR773726 late HIS2Hp 16S ribosomal RNA gene, partial (D098898 1.1 Zema II D SIHF08 780 99% with Uncultured Fraukia sp. iso­ FR773727 (Q8) late HIS2Hp I6S ribosomal RNA gene, partial (DQ98898 1.1 Below La- SIHF09 730 95% with Uncultured Fraukia sp. iso­ FR773728 chen late HIS2Hp 16S ribosomal RNA (Q 18) gene, partial (D098898 1.1 Zema l+ SIHF!O 797 95% with Uncultured Fraukia sp. iso­ FR773729 (QIO) late HIS2Hp 16S ribosomal RNA gene, partial (D098898 1.1 Chako SIHFII 640 96% with Uncultured Fraukia sp. iso­ FR773730 River (Q20) late HIS2Hp 16S ribosomal RNA

*Q represents quadrates of table 4.5. clusters revealed the relatedness Similarly the second group comprises between the II H. salicifolia-Frankia HISF04 and HISF09 with genetic studied. Cluster I consists of 7 isolates, proximity of 2.85792. Cluster II which is the larger cluster compared to comprises HISF07, HISF!l, HISF08 the other one, which consists of 4 and HISFlO with their genetic isolates. Cluster I can be divided into proximity given in fig. 4.16. two main groups: the first one Careful observation of the PCR-RFLP comprising HISF02, HISF03, HISF05 dendrogram and its correlation with the and IDSF06 with genetic proximity or table 4.5 (for quadrates, geographical similarity measures of 0.0000. conditions and the altitude from where RESULTS AND DISCUSSION 117 the amples were collected) and table proximity. This sample was collected 4.22, it i clearly understood that the at an altitude of l0206ft. Except for the Frankia i olates belonging to similar or altitudinal differences in the second near to imilar altitude have formed a group of clu ter I [HISF04 (9960ft) and close proximity. The altitude of group HISF09 (8450ft)], almost aJI have one of cluster I ranges between 8299- shown close proximity based on close 88 14ft. Similarly the altitude of c luster altitudinal ranges. The second group of II i olate ranges from 8937-9960ft. clu ter I indicate that apart from Similarly in the dendrogram it can also altitude, some other factor may also be een that HISFO1 howed a genetic affect the genetic diver ity of Frankia. proximity of 24.87490 and is placed far PCR based 16S rDNA-RFLP tudies away from other isolate of close have been u ed in eco-biotechnological

Figure 4.15: Restriction digestion gels o f PCR amplified 16S rRNA Frankia DNA of H. sa/icifolia u ing different enzymes. Lane ML= I kb molecular ladder (A.DNA), Lane A-K=SIHFO 1-SIHF II ( I I isolates). A=Restriction digesti on using A lul , B=Re tricti on digestion using Haelll, C=Restriction digestion u ing Mspl , and D=Restriction digestion u ing Hpall RESULTS AND DISCUSSION 118

2~4~. RB77 44 Qr90,------H IS F 01 +H I SF02 +-1 r l2 +HISF03 -101 +.;17:-. -;:-:50:::-::2:-:6:-::-1---3 l HI SFOS 6,-. -=-7 s=-4.,....,8:-::7-----6 l HI SF06 r +~-----2 ~ 857 92 HISF04 ~1 0.6174!~~~ ------9 4.64469 2 ~ 85? 92 H I S F09 HI SF07 2 4 82 79.11608 r8 +;9" .' 1"'1' 6:;:;0:;::8-----HI SFll 3 I +5I 5,...' 8:;;:8"'9"1..,.5--HI SF08 ~. 95175 I HISF10 5.88915

Figure 4.16: Dendrogram based on Nei's ( 1972) Genetic distance: Method=UPGMA modified from NEIGHBOR procedure of PHYLIP Version 3.5 studie as a procedure for studying the means in understanding how abiotic diversity of strains isolated from nature factors like temperature, precipitation, (Schraft et al., 1996; Urakawa et al. , seasonality (Graham et al., 2004; 1997; Yallaeys et al., 1997). Wiens and Graham, 2005), aspect,

In this study it has been found that, elevation, slope, etc. influences the apart from various other factors geographic limits of lineages and discussed above and in the foregoing species. To address the idea I have made an approach to use remote chapters, environmental conditions sensing satellite FCC data along with (elevation, aspect, slope), soil composition, geographic and Survey of India maps (both 1:50000 mkroclimatic conditions of Lachen scale) in generating an environmental map (land use & land cover, slope, Valley of North Sikk.im have been found to play a major role genetic elevation and aspect) to study the diversity of Frankia species found m environmental factors of H. salicifolia the root nodules of H. salicifolia. growing area. Land use and land cover data proves especially valuable for 2004; Elith et al., 2006). The use of predicting the distribution of both such kind of database in envi ronmental individual species (Jennings, 2000) and niche modelling (Soberon and assembly of different species (Kerr et Peterson, 2005) with relation to the a/. , 200 1) across broad areas that could species is proving to be a powerful not otherwi se be surveyed because of RESULTS AND DISCUSSION 119 the tough and some inaccessible microbes directly. The environmental terrains ofthe study area. factors act as a gene controlling

Variations in environmental factors enzyme system (McArthur et al., like nutrient concentration, nutrient 1988). Therefore, there must be a availability, temperature, pH, etc. have relationship between genetic diversity been found to affect the kinetics of soil of Frankia populations with their

Table 4.23: Sequences used in phylogenetic analysis with their sequence abbrevia­ tions, description and GenBank accession numbers Sequence code Description GenBankac. (GenBank) No. SIHFO!f Uncultured Franlda sp. partia116S rRNA gene from Hippo- FR773720 phae salicifolia root nodule isolate SIHF06f -do- FR773725 SIHF04f -do- FR773723 SIHF03f -do- FR773722 SIHF02f -do- FR773721 SIHF09f -do- FR773728 SIHF05f -do- FR773724 SIHF!lf -do- FR773730 SIHF07f -do- FR773726 SIHF08f -do- FR773727 SIHF10f -do- FR773729 EAN1PEC Frankia sp., complete genome CP000820 Eill1C Frankia sp., complete genome CP002299 EUN1F Franlda sp. partial 16S rRNA gene, Sn5-8 isolate AI408875 FDG Frankia sp. 5S, 16S, and 23S ribosomal RNA genes, com- M55343 plete sequences. CCI3 Frankia sp. Complete genome. · CP000249 ACN Frankia alni str. ACN14A chromosome, complete sequence. CT573213 NOC Nocardia farcinica IFM 10152 DNA, complete genome. AP006618 SAC Saccharopolyspora erythraea NRRL2338 complete genome AM420293 THER Thermomonospora curvataDSM 43183, complete genome. CP001738 XYL Xylanimonas cellulosilyticaDSM 15894, complete genome. CP001821 GEO Geodermatophilus obscurusDSM 43160, complete genome. CP001867 KRIB Kribbella flavida DSM 17836, complete genome. CP001736 NOCD Nocardioidessp.JS614, complete genome CP000509 NAK Nakamurella multipartite DSM 44233, complete genome. CP001737 KOC KocuriarhizophilaDC2201 DNA, complete genome. AP009152 MICR Micrococcus luteusNCTC 2665, complete genome. CP001628 BEU Beutenbergia cave mae DSM 12333, complete genome. CP001618 CLAVIS Clavibacter michiganensis subsp. Sepedonicus, complete AM849034 genome. CLAVIM Clavibacter michiganensis, partial 16S rRNA gene, strain P AI310416 250/01. STCOE Streptomyces coelicolo rA3(2) complete genome; segment AL939108 5/29. STAY Streptomyces avermitilisMA-4680 DNA, complete genome BA000030 STGR Streptomyces griseus subsp. Griseus NBRC 13350 DNA, AP009493 complete genome. RUB Rubrobacter xy1anophilus partial 16S rRNA gene. AI243871 RESULTS AND DISCUSSION 120

Table 4.24: Genetic polymorphism of Frankia detected by RFLP marker by various enzymes Enzymes used Bands generated Polymorphic bands %age of polymorphism Alul 3 2 66.6 Haem 6 3 50 Hpall 7 5 71.42 Mspl 2 I 50 Total 18 11 61.11

0 environmental conditions. Studies have also been conducted on microbial populations to show close relation 0 0 ~ between the genetic diversity and ~ 00 Cl) 0 environmental changes of a particular 0 ecosystem (Nevo et al., 1984) and some evidences have proved positive

0 relation between genetic polymorphism 00 "'- <-00 "' "' - 00 and environmental heterogenecity Cl)~ Odd "'-- (Hedrick et al., 1976). The Frankia 0 ('l"'.a 00 C"'l C'l"'' ...-~r-..-.N population diversity has been shown to V":l...-IV"lOO N-N- dddd be affected by ecological factors determined by elevation, like temperature, precipitation, etc. and soil

0 0 composition parameters like pH, OC, moisture, type, etc. (Chen et al., 2008).

As has already been discussed m prevrous chapters, H. salicifolia are good sources of dietary supplement, OOOOO'<:;f-C""'('f')-.:::TC'I"'' t- J.ONMV"l...-..! ...... -i NOOOOC'ltn M,...... ,...... C"'l N natural antioxidants including c:i ciOc:icici carotenoids, vitamins, phenols, OOOOOOVMM-.::t("') t- VlNNl/1- ...... NOOOONV"' flavonoids, and a high level of ,...... C""'',...... ,...... ("") N d cidddd antioxidative property against free radical species. The total antioxidant activity can vary from plants growing in different environmental conditions, and these may affect overall protective benefits of human health, which needs RESULTS AND DISCUSSION 121 to be further investigated. Antioxidant providing the antioxidative property of study from the leaf and bark of male H. salicifolia of Sikkim has opened up and female plants of H. salicifolia is a new scope for using these compounds the first report from Sikkim Himalayas to correlate the symbiotic interaction of but there may not be any report on host-microorganism and genetic antioxidant (phenolics, flavonoids, etc.) diversity of Frankia to get a better of the host plant affecting genetic cultivar of this ecologically and diversity of Frankia in Hippophae. economically important plant species.

It has been understood that during Quantification of root nodules from H. biotic interactions, plant secondary salicifolia plants growing in different metabolites and phenolics give positive locations of the study area can be signals, during plant-environment attributed to assessing the population interactions. One such interaction is size of Frankia in the study area. Jeong during the early stage of nodule and M yrold (2003) have also stated formation and establishment of that counting of nodule from different symbiotic relationship, phenolic based actinorhizal plants and can be the reorganisation mechanism between the indirect means of measurement of host and bacterial symbiont is involved population size of host infective (Gould and Lister, 2006). These signal Frankia population in the soil. interaction mechanism is well studied Vegetative propagation of H. in mycorrhiza (Steinkellner et al., salicifolia using different means like 2007) and legumes (Cooper, 2004) but cuttings using different combinations very little is known about the role of and proportions of growth regulators phenolics in the symbiosis between and layering m different soil actinorhizal plants and Frankia, and no combinations was studied. Further attempts have even been made to study propagation through seeds m this type of signal interactions in combination with different chemicals Hippophae salicifolia. There has been to study breaking of dormancy was a report on the responsiveness of carried out in this study. The species Frankia strains to Myrica gale fruit showed its survival and adaptability in exudates phenolics with their symbiotic a different condition from its natural compatibility by Popovici et al. (20 I 0). habitat. The outcome of this study may However, my study apart from not be directly involved in the study of RESULTS AND DISCUSSION 122 genetic diversity but the outcome of and function m actinorhizal plants this can be useful in planning, getting (Huss-Danel11997). In my study also it desired selected plant accession and was revealed that the roots of H. faster propagation compared to natural salicifolia growing in the riverine areas condition. The root nodules out of possessed more nodulations compared these propagated plants could have to non riverine areas. However, the given an interesting result, if Frankia difference in diversity observed could could have been isolated from them in be understood by the characteristics of pure culture. Insufficient nodules per the niches occupied by the plant, which plant for genomic DNA isolation in turn controls its microsymbiont in hindered study of genetic diversity due the root. Therefore we can conclude to enviromnental and time factor to that environmental factors control the complete this research work, which genetic diversity of Frankia in the root will be taken up in future. nodules of the actinorhizal plant and H.

During the study of genetic diversity of salicifolia carmot be an exception. Frankia associated with Hippophae of Earnest beginning in the study of Lachen, I took up the environmental microbial aspect of Frankia species parameters of the study area, which can started only 15 years ago when first also reflect the plant-environment strain was armounced. It was also relationship on the genetic diversity of found that Frankia were slow growing Frankia through the host. Intense organisms and much of intervening studies have revealed that host plant time has been spent on establishment controls the root nodulation and of different techniques of growing nitrogen fixation processes m Frankia and pursuing hypothesis actinorhizal plants (Dobritsa and related to the nature of organism, its Novik, 1992; Wall and Huss-Danell, vesicle, spores and physiology evolved 1997; Valverde and Wall, 1999). from the past studies. If we see from Enviromnental factors like light, water, the global perspective, fair amount is soil nutrients availability, soil pH, etc. known about Frankia strains on and bacterial factors such as phylogenetic position, structure and physiologic state, population and function of spores and vesicles, ability to fix nitrogen are also thought physiology of carbon and nitrogen to effect nodule development, growth metabolism, ecology in the soil and RESULTS AND DISCUSSION 123 nodule. Recently some details have actinorhizal genera described (Benson started pouring in about the genetics of and Silvester, 1993), 11 genera this unique microorganism. In my fulfilling Koch's postulate could have study, I also isolated Frankia strains of their 16S rDNA sequence determined H. salicifolia in pure culture, which and compared for its diversity study may also be the first isolation from the (Normand et al., 1996).

Hills of Sikkim. The same has been For the presence or absence of reflected in t!Ie results and discussion actinorhizal plants, climatic and soil chapter (chapter IV). This has led to conditions are t!Ie factor that always t!Ie understanding of the structure of influences Frankia (Dawson et al., this isolated strain and also helped in 1989; Huguet et al., 2004; Oakley et conducting plant infectivity test. The al., 2004). This study focuses on single amount of Frankia colonies could not Frankia-nodulating species growing in suffice the isolation of genomic DNA variable microclirnatic region at from pure culture, hence genomic different elevations, aspects and slopes DNA for its genetic diversity was of Lachen valley. Total of 11 DNA isolated from the root nodules directly, isolates gave positive results when as has been done in many cases of such subjected to PCR. Study of genetic studies in various actinorhizal plants. diversity using H. salicifolia-Frankia During t!Ie infectivity test in vitro and genome and comparing it wit!I oilier in situ t!Ie Hippophae plants showed genome sequences having 16S rRNA, positive results by getting infected with and also PCR-RFLP study, provided the Frankia inoculums. This has not some interesting facts related to genetic only showed that Hippophae-Frankia diversity of Frankia strains of H. strains are spore+ but has also opened salicifolia occupying this niche wit!I up an area to carry out more work to physical! y untraceable climatic study communication mechanism conditions. It is understood t!Iat between t!Ie actinomycetes and the host distribution of this Frankia strain is actinorhizal plants in the rhizosphere. also basically affected by altitudes or

Phylogenetic comparisons of Frankia the host plant growing habitat. Reports and its close lineages are promising by Dai et al. (2004); lgual et al. (2006); approach to understand t!Ie evolution Jeong and Myrold (1999); Oakley et al. of t!Ie organisms. Out of 21 (2004) and few others supports this RESULTS AND DISCUSSION 124 findings that topography and altitude the world on evolution and genetic plays a vital role in composition of diversity of Frankia (in the soil or in Frankia community. Soil in the study different actinorhizal hosts or m area was almost the same throughout, different ecological conditions so not much difference was observed involving different ecological and due to soil conditions or their environmental parameters), many compositions. Although some authors opportunities to study this organism are like Burleigh and Dawson (1994); coming up. It is felt that there are still Crannell et al. (1994) have co-related more potential working areas in the that soil pH, organic matters, etc. may days to come, which include studies on influence Frankia genotypes but from genetics and molecular genetics of the various studies and results expressed in host plant infection process, symbiotic chapter IV, especially with reference to interaction, the phylogeny and Frankia, even soil pH or organic taxonomy of those strains that have yet matters, had no major role to play in to yield infective strains and Frankia population and distribution in physiological studies in relation to Lachen valley. Some ecological factors symbiosis. Above all, there is a great like altitude, aspect, slope and need to understand integrated microclimatic conditions like phylogenetic and taxonomic studies of temperature and moisture in the H. Frankia strains to understand its actual salicifolia growing area have shown phylogeny and taxonomy, which will the genetic diversity among its Frankia lead to universal identity of this strains, perhaps through host or direct microorganism. Presently, out of many impact, and also may be the major phylogenetic studies carried out by determinants of Frankia population in researchers globally, the outcome is the study area. Similar work based on based either on a particular host, region Frankia population diversity on H. or environmental factor. Very few rhamnoides taking few ecological comparisons have been found, which is parameters focussing on elevation and not sufficient to present the actual plant cover by Chen et al. (2008) status of Frankia strains on a global supports my findings. basis. The work is difficult and time

With the advent of many innovative consuming too, but such genetic and informative researches throughout studies would help to lay a base with a RESULTS AND DISCUSSION 125 unifying hypothesis to answer changes, which are proposed to occur questions like why actinorhizal nodules in the next few decades. Since these form on certain distantly related plants species can survive and ameliorate the but not on other plants of the same effect of vegetation losses because of family. This may share co-evolution nutritional deficiency in the soil, they theory of both the symbionts and their can be a pioneering species to keep the diversity. vegetation integrity of the area but its

Broadly, actinorhizal plants play an practical issue of actinorhizal important role in different symbiosis needs to be addressed along with genetic diversity, which will not environmental conditions. Hippophae only explain its phylogeny and salicifolia growing in the Lachen valley of Sikkim too has been found to evolution but shall also help in occupy an important and influential understanding better variant of Frankia position in the existing ecosystem, strain for future planning of better eco­ especially in the context of climatic friendly cultivar. Conclusion

Integration of ecological and genetic sensing satellite FCC data along with approaches in molecular studies along Survey of India maps (both 1:50000 with geographic distributions of scale) in generating an environmental organisms is becoming more common map (land use & land cover, slope, (Johnson and Cicero, 2002; Lapointe elevation and aspect) to study the and Rissler, 2005). This is partly environmental factors of H salicifolia because of increasing amount and growing area. Land use and land cover accuracy in the process of collecting data proves especially valuable for socioeconomic and natural data, predicting the distribution of both housing fine-scaled climate data and individual species (Jennings, 2000) and new modelling techniques (Graham et assembly of different species (Kerr et a!., 2004; Elith eta!., 2006). The use of a!., 2001) across broad areas that could such kind of database in environmental not otherwise be surveyed because of niche modelling (Soberon and the tough and some inaccessible Peterson, 2005) with relation to the terrains of the study area. species is proving to be a powerful Variations in environmental factors means in understanding how abiotic like nutrient concentration, nutrient factors like temperature, precipitation, availability, temperature, pH, etc. have seasonality (Graham et a!., 2004; been found to affect the kinetics of soil Wiens and Graham, 2005), aspect, microbes directly. The environmental elevation, slope, etc. influences the factors act as a gene controlling geographic limits of lineages and enzyme system (McArthur et a!., species. To address the idea I have 1988). Therefore, there must be a made an approach to use remote CONCLUSION 127 relationship between genetic diversity the first report from Sikkim Himalayas of Frankia populations with their but there may not be any report on environmental conditions. Studies have antioxidant (phenolics, flavonoids, etc.) also been conducted on microbial of the host plant affecting genetic populations to show close relation diversity of Frankia in Hippophae. between the genetic diversity and It has been understood that during environmental changes of a particular biotic interactions, plant secondary ecosystem (Nevo et a/., 1984) and metabolites and phenolics give positive some evidences have proved positive signals, during plant -environment relation between genetic polymorphism interactions. One such interaction is and environmental heterogenecity during the early stage of nodule (Hedrick et al., 1976). The Frankia formation and establishment of population diversity has been shown to symbiotic relationship, phenolic based be affected by ecological factors reorganisation mechanism between the determined by elevation, like host and bacterial symbiont is involved temperature, precipitation, etc. and soil (Gould and Lister, 2006). These signal composition parameters like pH, OC, interaction mechanism is well studied moisture, type, etc. (Chen eta/., 2008). in mycorrhiza (Steinkellner et al., As has already been discussed in 2007) and legumes (Cooper, 2004) but prevwus chapters, H salicifolia are very little is known about the role of good sources of dietary supplement, phenolics in the symbiosis between natural antioxidants including actinorhizal plants and Frankia, and no carotenoids, vitamins, phenols, attempts have even been made to study flavonoids, and a high level of this type of signal interactions in antioxidative property against free Hippophae salicifolia. There has been radical species. The total antioxidant a report on the responsiveness of activity can vary from plants growing Frankia strains to Myrica gale fruit in different environmental conditions, exudates phenolics with their symbiotic and these may affect overall protective compatibility by Popovici et al. (20 10). benefits of human health, which needs However, my study apart from to be further investigated. Antioxidant providing the antioxidative property of study from the leaf and bark of male H salicifolia of Sikkim has opened up and female plants of H salicifolia is a new scope for using these compounds CONCLUSION 128 to correlate the symbiotic interaction of faster propagation compared to natural host-microorganism and genetic condition. The root nodules out of diversity of Frankia to get a better these propagated plants could have cultivar of this ecologically and given an interesting result, if Frankia economically important plant species. could have been isolated from them in pure culture. Insufficient nodules per Quantification of root nodules from H salicifolia plants growing in different plant for genomic DNA isolation hindered study of genetic diversity due locations of the study area can be to environmental and time factor to attributed to assessing the population size of Frankia in the study area. Jeong complete this research work, which and Myrold (2003) have also stated will be taken up in future. that counting of nodule from different During the study of genetic diversity of actinorhizal plants and can be the Frankia associated with Hippophae of indirect means of measurement of Lachen, I took up the environmental population size of host infective parameters of the study area, which can Frankia population in the soil. also reflect the plant-environment relationship on the genetic diversity of Vegetative propagation of H Frankia through the host. Intense salicifolia using different means like studies have revealed that host plant cuttings using different combinations controls the root nodulation and and proportions of growth regulators nitrogen fixation processes m and layering m different soil actinorhizal plants (Dobritsa and combinations was studied. Further Novik, 1992; Wall and Huss-Danell, propagation through seeds m 1997; Valverde and Wall, 1999). combination with different chemicals Environmental factors like light, water, to study breaking of dormancy was soil nutrients availability, soil pH, etc. carried out in this study. The species and bacterial factors such as showed its survival and adaptability in physiologic state, population and a different condition from its natural ability to fix nitrogen are also thought habitat. The outcome of this study may to effect nodule development, growth not be directly involved in the study of and function in actinorhizal plants genetic diversity but the outcome of (Huss-Danell 1997). In my study also it this can be useful in plarming, getting was revealed that the roots of H desired selected plant accession and CONCLUSION 129 salicifolia growing in the riverine areas study, I also isolated Frankia strains of possessed more nodulations compared H. salicifolia in pure culture, which to non riverine areas. However, the may also be the first isolation from the difference in diversity observed could Hills of Sikkim. The same has been be understood by the characteristics of reflected in the results and discussion the niches occupied by the plant, which chapter (chapter IV). This has led to in turn controls its microsymbiont in the understanding of the structure of the root. Th~;refore we can conclude this isolated strain and also helped in that environmental factors control the conducting plant infectivity test. The genetic diversity of Frankia in the root amount of Frankia colonies could not nodules of the actinorhizal plant and H. suffice the isolation of genomic DNA salicifolia cannot be an exception. from pure culture, hence genomic DNA for its genetic diversity was Earnest beginning in the study of isolated from the root nodules directly, microbial aspect of Frankia species as has been done in many cases of such started only 15 years ago when first studies in various actinorhizal plants. strain was announced. It was also found that Frankia were slow growing During the infectivity test in vitro and organisms and much of intervening in situ the Hippophae plants showed time has been spent on establishment positive results by getting infected with of different techniques of growing the Frankia inoculums. This has not Frankia and pursumg hypothesis only showed that Hippophae-Frankia related to the nature of organism, its strains are spore+ but has also opened vesicle, spores and physiology evolved up an area to carry out more work to from the past studies. If we see from study communication mechanism the global perspective, fair amount is between the actinomycetes and the host known about Frankia strains on actinorhizal plants in the rhizosphere. phylogenetic position, structure and Phylogenetic comparisons of Frankia function of spores and vesicles, and its close lineages are promising physiology of carbon and nitrogen approach to understand the evolution metabolism, ecology in the soil and of the organisms. Out of 21 nodule. Recently some details have actinorhizal genera described (Benson started pouring in about the genetics of and Silvester, 1993), II genera this unique microorganism. In my fulfilling Koch's postulate could have CONCLUSION 130 their 16S rDNA sequence determined area was almost the same throughout, and compared for its diversity study so not much difference was observed (Normand eta/., 1996). due to soil conditions or their compositions. Although some authors For the presence or absence of like Burleigh and Dawson (1994); actinorhizal plants, climatic and soil Crannell et a/. (1994) have co-related conditions are the factor that always that soil pH, organic matters, etc. may influences Frankia (Dawson et a/., influence Frankia genotypes but from 1989; Huguet et al., 2004; Oakley et various studies and results expressed in al., 2004). This study focuses on single chapter IV, especially with reference to Frankia-nodulating species growing in Frankia, even soil pH or organic variable microclimatic region at matters, had no major role to play in different elevations, aspects and slopes Frankia population and distribution in of Lachen valley. Total of 11 DNA Lachen valley. Some ecological factors isolates gave positive results when like altitude, aspect, slope and subjected to PCR. Study of genetic microc!imatic conditions like diversity using H salicifolia-Frankia temperature and moisture in the H genome and comparing it with other salicifolia growing area have shown genome sequences having 16S rRNA, the genetic diversity among its Frankia and also PCR-RFLP study, provided strains, perhaps through host or direct some interesting facts related to genetic impact, and also may be the major diversity of Frankia strains of H determinants of Frankia population in salicifolia occupying this niche with the study area. Similar work based on physically untraceable climatic Frankia population diversity on H conditions. It IS understood that rhamnoides taking few ecological distribution of this Frankia strain is parameters focussing on elevation and also basically affected by altitudes or plant cover by Chen et al. (2008) the host plant growing habitat. Reports supports my findings. by Dai et al. (2004); Igual eta/. (2006); Jeong and Myrold (1999); Oakley eta/. With the advent of many innovative (2004) and few others supports this and informative researches throughout findings that topography and altitude the world on evolution and genetic plays a vital role in composition of diversity of Frankia (in the soil or in Frankia community. Soil in the study different actinorhizal hosts or in CONCLUSION 131 different ecological conditions unifYing hypothesis to answer involving different ecological and questions like why actinorhizal nodules environmental parameters), many form on certain distantly related plants opportunities to study this organism are but not on other plants of the same coming up. It is felt that there are still family. This may share co-evolution more potential working areas in the theory of both the symbionts and their days to come, which include studies on diversity. genetics and molecular genetics of the Broadly, actinorhizal plants play an host plant infection process, symbiotic important role Ill different interaction, the phylogeny and environmental conditions. Hippophae taxonomy of those strains that have yet salicifolia growing in the Lachen to yield infective strains and valley of Sikkim too has been found to physiological studies in relation to occupy an important and influential symbiosis. Above all, there is a great position in the existing ecosystem, need to understand integrated especially in the context of climatic phylogenetic and taxonomic studies of changes, which are proposed to occur Frankia strains to understand its actual in the next few decades. Since these phylogeny and taxonomy, which will species can survive and ameliorate the lead to universal identity of this effect of vegetation losses because of microorganism. Presently, out of many nutritional deficiency in the soil, they phylogenetic studies carried out by can be a pioneering species to keep the researchers globally, the outcome is vegetation integrity of the area but its based either on a particular host, region practical issue of actinorhizal or environmental factor. Very few symbiosis needs to be addressed along comparisons have been found, which is with genetic diversity, which will not not sufficient to present the actual only explain its phylogeny and status of Frankia strains on a global evolution but shall also help in basis. The work is difficult and time understanding better variant of Frankia consuming too, but such genetic strain for future planning of better eco­ studies would help to lay a base with a friendly cultivar. References

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Basistha, B. C. and I. M. Adhikari (2003). Response of Physiologically Active Substance in the Germination of Sea buckthorn (Hippophae L). Journal of Hill Research. 16: 99-100.

Basistha, B. C.; N. P. Sharma, L. Lepcha, M. L. Arrawatia and A. Sen. (2009). Ecology of Hippophae salicifolia D. Don of temperate and sub-alpine forests of North Sikkim Himalayas-a case study. Symbiosis. 1-2 (50): 87-95.

Basistha, B. C.; N. P. Sharma and A. Sen (2009). Understanding-Agro techniques of Hippophae salicifolia D. Don (Sea buckthorn) of Sikkim Himalayas. Proceedings of the Fourth International Sea buckthorn Association Conference., Belokuriha (Altai Region), Russia. pp. 77-78.

Goyal, A. K; B. C. Basistha, A. Sen and S. K. Middha (2011). Antioxidant profiling of Hippophae salicifolia D. Don growing in sacred forests of Sikkim, India. Communicated to Journal of Functional Plant Biology.

Basistha, B. C. and A. Sen (2011). Sea buckthorn and its microsymbiont-a review. NBU Journal of Plant Sciencec. 1(5): 67-84. APPENDIX-I

Composition of Frankia culture media used during work.

DPM-Nitrogen free medium. (Baker and 0' Keefe, 1984) Constituents Stock solution Working solution (Per liter) (Per liter)

[A] Macro-nutrients lOX

KH2P04 1 lOg 734 !1M MgS04.?H201 l.Og 0899 !1M 2 CaCh.ZH20 O.lg 0068 !1M 5 C3Hs02Na (Sod. Propionate) 12.0g 12490 !1M

[B] Micro-nutrients lOOOX

H3BO/ 2.860g 46.3 !1M MnCh.4H201 1.810g 9.1 J.LM ZnS04.?H201 0.220g 0.8 J.LM CuS04.SH201 0.080g 0.3 !1M Na2MoO/ 0.025g 0.1 J.LM 2 CoCh.?H20 0.025g 0.1 J.LM

[C] Iron lOOOX

FeSo4. ?H202 10.008g 370.4!JM 3 Na2EDTA 13.410 36.0 J.LM

PH-7.0

1E MERCK (India) Ltd. Mumbai, India. 2SRL SISCO RESEARCH LABORATORIES Pvt. Ltd. Mumbai, India. 3QUALIGENS Fine Chemicals, Glaxo India Ltd. Mumbai, India. 4HI MEDIA Laboratories Limited, Mumbai, India. 5SIGMA-ALDRICH Chemicals Pvt. Ltd. USA.

AI Q-mod Medium {Lalonde and Calvert, 1979) Constituents Stock solution Working solution (Per liter) (Per liter)

[A] Macro-nutrients lOX

1 KzHP04 3.0g l722~M 1 NaHzP04.2Hz0 2.3g 1474~M 1 MgS04.7Hz0 2.0g 0811~ 1 KC1 2.0g 1682~M 4 D-Glucose lOOg 55506~ Yeast Extrace 0.5g Peptone3 50.5g

[B] Micro-nutrients lOOOX

H3BO/ l.500g 24.3~ 1 MnS04.HzO 0.800g 4.7~ 1 ZnS04.7H20 0.600g 2.1~ 1 CuS04.5H20 O.lOOg 0.4~ 1 (NH4) s Mo-,0 244Hz0 0.200g 0.16~ 2 CoS04.1Hz0 0.025g 0.09~

[C] Iron lOOOX

2 CsHs07. Hz0 lO.Og 47.6~ 2 CsHs07 Fe. 3Hz0 lO.Og 33.5~

[D] Lipid supplement lOX

4 L- oc - Lecithin 0.400g pH-7.0

1E MERCK (India) Ltd. Mumbai, India. 2SRL SISCO Research Laboratories Pvt. Ltd. Mumbai, India. 3HI MEDIA Laboratories Limited, Mumbai, India 4SIGMA-ALDRICH Chemicals J>vt. Ltd. USA.

A2 Hoagland Medium N2-free (modified)

Constituents Stock solution Working solution (Per liter) (Per liter)

[A] Macro-nutrients lOX

1 KHzP04 1.36g 999~-tM 1 MgS04.?Hz0 3.38g 30391-LM

[8] Micro-nutrients lOOOX

H3BO/ 2.860g 46.31-LM 1 MnCh.4HzO 1.810g 9.11-LM 1 ZnS04.?Hz0 0.220g 0.81-LM 1 CuS04.SHz0 0.080g 0.3 11M NazMoO/ 0.025g 0.11-LM 2 CoCh.7Hz0 0.025g 0.1~-tM

[C] Iron lOOOX

2 FeS04. 7Hz0 10.008g 370.41-LM 3 NazEDTA 13.410g 36.0 11M pH -7.0

1E MERCK (India) Ltd. Mumbai, India. 2SRL SISCO RESEARCH LABORATORIES Pvt. Ltd. Mumbai, India. 3QUALIGENS Fine Chemicals, Glaxo India Ltd. Mumbai, India.

A3 BAP medium (modified from Fontaine et al., 1986)

Co11stitue11ts Co11ce11tratio11

[A] Macro-nutrients grams/liter

K2HP041 0.592 KH2PO/ 0.953 MgS04.?H201 0.0493 Nli!Cl1 0.267 CaC1z.2H202 0.010 Sodium Propionate4 0.480

[B] Micro-nutrients grams/liter

CoS04.?H202 0.000001 CuS04.SH201 0.00008 H3BO/ 0.00286 MnClz,4H201 0.00181 NaMo04.2H202 0.000025 ZnS04.?H201 0.00022

[C] Iron grams/liter

FeNa-EDTA2 0.013

[C] Vitamins milligrams/liter

Biotin3 0.0225 Thiamine Hce 0.01 Nicotinic acid 3 0.05 Folic acid2 0.01 Pyridoxine HC12 0.05 Calcium Pantothenate3 0.01 Riboflavin3 0.01 pH-6.7

1MERCK (India) Ltd. Mumbai, India. 2SRL SISCO RESEARCH LABORATORIES Pvt. Ltd. Mumbai, India. 3HI MEDIA Laboratories Limited, Mumbai, India 4SIGMA-ALDRICH Chemicals Pvt. Ltd. USA

A4 Frankia Medium (after Benson, 1982)

Constituents Concentration

[A] Macro-nutrients Grams per liter

CaCO/ 0.05 FeNa-EDTA2 0.16 KzHPO/ 2.0 KzHPO/ 3.0 MgS04.?Hz01 0.2 NaC11 0.3

[B] Micro-nutrients Milligram per liter

CuS04.SHz01 0.04 H3B03 1 1.5 MnS04.Hz01 4.5 NazMo04.Hz02 0.25 ZnS04.?Hz01 1.5 Cassamino Acid3 3000 Na-Pyruvate3 3000

[C] Vitamins Milligram per liter

Nicotinic acid 3 0.5 Pyrimidine HCI2 0.5 Thiamine Hce 0.1 pH-7

1E MERCK (India) Ltd. Mumbai, India. 2SRL SISCO Research Laboratories Pvt. Ltd. Mumbai, India. 3HIMEDIA Laboratories Limited, Mumbai, India.

AS APPENDIX-II

Composition of Buffers

=> 0.2 M Cacodylate buffer (1 X concentration)

197 mM Cacodylic acid. [Dimethylarsinic acid [Na (CH3)zAs0z.3HzO] Sodium Salt1

Sodium cacodylate (42.2g) was dissolved in 800ml of sterile water. pH adjusted to 7.4 with HCl and volume adjusted to IOOOml with sterile double distilled water.

1Sigma chemical Co USA

A6 DNA Extraction ( C-TAB) Buffer (1 X concentration)

100 mM Trizma base3 (pH-8.0 at 27°C) 20mM EDTA2 (pH-8.0) L4MNaC11 2% (WN) CTAB3 2% (WN) pypp3 54g of molecular biology grade Trizma base [Sigma, USA. Cat No. T-1503, Tris

(hydroxymethyl) arninomethane, C4HuN03. (M.W. 12.llg) was dissolved in 800ml of sterile double distilled water; pH adjusted to 8.0 and was divided to into two parts. To one part 7.44g EDTA was added and in other part 8L82g NaCl, 20g CTAB

(Hexadecyl trimethyl ammonium bromide, C19~2NBr) and lOg PVPP was added. Both the parts were then mixed.

1MERCK (India) Ltd. Mumbai, India. 2Qualigens Fine Chemicals, Mumbai, India. 3SIGMA-ALDRICH Chemicals Pvt. Ltd. USA 4SRL Chemicals, Mumbai, India

-- ~

A7 DNA loading Buffer (6 X Concentration) (Type III, Sambrook et al, 2001)

0.25% Bromophenol blue1 0.25% Xylene cyanol FF2 30% Glycerol3 in Double Distilled water.

2.5g of Bromophenol blue and Xylene cyanol was dissolved in lOOOml of 30% Glycerol.

1E MERCK (India) Ltd. Mumbai, India. 2S D Fine Chemicals, India. 3Qualigens Fine Chemicals, Mumbai, India.

PBS-Phosphate Buffered Saline (pH-7.4)

137mMNaCl1 2.7 mMKCl1 10 mM NazHPOi 1.8 mM KHzP04 1

8g of NaCl, 0.2g of KCl, 1.44g of Na2HP04. and 0.24g of KH2P04 were dissolved in 800ml of sterile double distilled water and final volume was made up to 1litre with sterile double distilled water. The pH was adjusted to 7.4 with HCJ.

1EMERCK (India) Ltd. Mumbai, India.

AS Peeling Buffer

NaC11 8g KC1 1 0.2g NazHPO/ 1.44g KHzP04 I 0.24g

The above components were dissolved in 1 litre of double distilled water and pH adjusted to 7.4 with HC!. Aft~r pH adjustment, 30g of polyvinyl pyrollidone (PVP) 2 was added and autoclaved.

1EMERCK (India) Ltd. Mumbai, India. 2SIGMA-ALDRICH Chemicals Pvt. Ltd. USA

=> TE-Tris EDTA Buffer (pH-S)

10 rnM Tris (pH-8) 10 rnM EDTA (pH-8) 1.2lg of molecular biology grade Trizma base (Sigma, USA Cat No. 1503, Tris

(hydroxymethyl) aminomethane, C4H 11 N03, (MW-12.1g) was dissolved in 400ml of double distilled water and pH adjusted to 8.0 with cone. HC11 and autoclaved. Similarly, 0.732g Di-sodium EDTA2 was dissolved in 400m1 of double distilled water. The solution was stirred properly and pH was adjusted to 8.0 with NaOH1 pellets and sterilized by autoclaving. Both the solutions were then mixed and volume made up to 1 liter by adding sterilized double distilled water.

1EMERCK (India) Ltd. Mumbai, India. 2Qualigens Fine Chemicals, Mumbai, India.

A9 => TBE-Tris-Borate EDT A Buffer (5 X concentration)

0.045 M Tris-Borate 0.001 MEDTA

Preparation of 5 X stock

54g of molecular biology grade Trizma base (Sigma, USA Cat No. 1503, Tris 1 (hydroxymethyl) aruinomethane, C4Hl!N03, (MW-!.21g) and 27.5g Boric acid were dissolved in 800ml of sterile double distilled water. To it 20m! of 0.5 M EDTA2 (pH- 1 8.0) was added. pH was adjusted to 7.6 with cone. HC1 . The solution was dispensed into aliquots and kept at room temperature. TBE was used in a final concentration of 1 X, by diluting 5 X stock to 1 X by adding Double Distilled HzO.

1E MERCK (India) Ltd. Mumbai, India. 2Qualigens Fine Chemicals, Mumbai, India.

=> DNA Wash Buffer (1 X concentration)

0.02 M Tris. Cl (pH- 7.2) 2mM EDTA (pH-8.0) O.OlMNaCl Supplied as a part of DNA cleaning kit.

AlO PCR buffer (1 X concentration)

I OOmM Tris. HCl pH 8.3 (at 25°C) 500mM KCl 15mM MgCI2 0.01 % (W/V) gelatin. Supplied with Hi-Media PCR kit, DNA polymerase as 10% concentrated stock and recommended for use with the same.

A ll