WNRF Technical Bulletin : 12 Study on ecotypic, morphological and genetic variations in accessions of Morinda citrifolia and its allies from varied eco-climatic habitats in Tamil Nadu using molecular tools

Dr. S John Britto SJ Director The Rapinat Herbarium and Centre for Molecular Systematics St. Joseph's College (Autonomous), Tiruchriappalli 620 002, Tamil Nadu, India

World Noni Research Foundation 12, Rajiv Gandhi Road, Perungudi, Chennai – 600 096, India Phone : 91-44-490 11 111 Fax : 91-44-490 11 149 E mail : [email protected] Visit : www.worldnoni.net

October, 2013 Published by : Prof. P. I. Peter Chairman Noni BioTech, Chennai, India

Edited by : Dr. P. Rethinam Dr. T. Marimuthu Dr. K.V. Peter

Author Dr. S John Britto SJ

Citation John Britto, S. 2013. Study on ecotypic, morphological and genetic variations in accessions of Morinda citrifolia and its allies from varied eco-climatic habitats in Tamil Nadu using molecular tools. WNRF Technical Bulletin-12, World Noni Research Foundation, India. p. 60

Printed at : Reliance Printers 9, Sardar Patel Road, Adyar, Chennai - 600 020 Contents

No. Particulars Page No.

Foreword i

About the Project ii

Preface iii

1. Introduction 1

2. State of knowledge 3

3. Objectives 9

4. Experimental details 10

5. Experimental findings 20

6. Summary and conclusion 45

References 47

Plates 54 i

Foreword

The World Noni Research Foundation Chennai funds research projects on Noni in five areas of scientific interest, Improvement, protection, clinical and pharmacological research and food science. Rev.Fr. Dr. S. John Britto was supported to conduct research on "Study on ecotypic, morphological and genetic variations in accessions of Morinda citrifolia L. and its allies from varied eco-climatic habitats in Tamil Nadu using molecular tools". All the technical programmes envisaged in the project were conducted well and the results are well presented. Results comprised analysis of the field data with description of over 1250 ecotypes of noni along with charts and photographs; antimicrobial activities of ecotypes and photochemistry of selected accessions; molecular systematic including bar coding. A herbarium of all the ecotypes is made available in duplicate. Digitization of herbarium collection is in progress. The project identified several promising lines for collection and further use. The technical bulletin adds to the wealth of knowledge on Noni.

I congratulate Rev. Fr. Dr. S. Britto for this outstanding research work.

Chennai - 600 096 (Kirti Singh) October, 2013 Chairperson ii

About the Project

The project "Study on ecotypic, morphological and genetic variations in accessions of Morinda citrifolia and it's allies from varied eco-climatic habitats in Tamil Nadu using molecular tools" was conceptualized after scientific deliberations and meticulously executed. It is the first time a herbarium inclusively for Morinda citrifolia ecotypes and their allied species was prepared in duplicate and one set deposited at World Noni Research Foundation Chennai. It is also the first time a bar coding was done on species of Morinda. Elite types of Noni were identified and located for future collection.

Antimicrobial activities of Noni fruit juice and leaf extracts were studied and the antibiotic properties established. Phytochemistry research on Noni fruits and leaves established anti-inflammatory and anti-diabetic properties. The veteran scholar conducted molecular systematics of Noni and it's allied species through RAPD and ISSR tools.

I place on record my highest level of appreciation for his invaluable research contribution to Noni Science. The efforts to prepare the technical bulletin from the completed project are appreciated.

Chennai - 600 096 (P. I. Peter) October, 2013 Chairman, Noni BioTech iii

Preface

It was with a sense of joy and challenge that I ventured on the Research Project on ‘Ecotypic, morphological and genetic variations in accessions of Morinda citrifolia and its allies from varied eco-climatic habitats using molecular tools’. As a result of this project a complete survey of Morinda citrifolia and its allies have been systematically studied. Besides the complete documentation of the taxonomic work based on strict taxonomic protocols, there is a record of field work data with exact locations of eco-climatic habitats with their latitude and longitude, geographic variations of collections sites and micro climatic data. Phenological variations have received the fullest attention during field work. To substantiate the above aspects several photographs have been taken. The most essential deliverable parts of the project are the carefully mounted herbarium specimens numbering around 1101 submitted to WNRF along with the copious Final Report. Technical programmes include the following: Accessions from different regions and habitats; Study of phenology and variation of phenotypes; Study of floral biology, fruiting pattern and natural regeneration; Antimicrobal studies on various accessions; Molecular systematic and Phytochemistry with GC-MS; Delimiting Accessions with promising characters; Submission of Final Report I am sure that the WNRF and ISNS will find the above not only invaluable but a Resource Base for further research along the applied aspects of Morinda citrifolia christened as the DIVINE NONI. I wish to express my sincere gratitude to World Noni Research Foundation International Society for Noni Science for the offer of a research project. It is a special privilege to thank Prof. P.I. Peter, Chairman Noni Bio Tech., Chennai, and Vice President ISNS. Dr. K.V. Peter, Director, WNRF, Chennai and Secretary of ISNS Dr. T. Marimuthu, Additional director WNRF, Chennai and Treasurer, ISNS and all the members of the executive council ISNS and the honorable members of Research Advisory Board WNRF and Heads of Research programmes, WNRF. I acknowledge with gratitude the assistance of my team members in Rapinat Herbarium, St. Joseph's College, Tiruchirappalli: A. Rajasekaran, T.R. Bhanumathi Thulasiraj, Hema Ramesh, A. Philominal Selvi, L. Lily, Dr. Sr. Seena, Dr. Sr. Jeslin Mr. J. Krishnakumar and M. Immanuvel Sahayaraj, N. Manimala and L. Joelri Michael Raj. I am sure that the WNRF and ISNS will appreciate this venture as a Resource Base for further research along the applied aspects of Morinda citrifolia christened as the DIVINE NONI.

Dr. S. John Britto SJ WNRF Technical Bulletin : 12 1

1. Introduction

Though Morindia citrifolia is a native of Queensland, it has become very much naturalised in all countries of South-East Asia from Indonesia to India. The vernacular name (English) Indian mulberry indicates its naturalness to the Indian subcontinent. As expected, it has established itself successfully in a variety of habitats and has probably hybridised with another local indigenous species such as Morindia pubescens. This is manifested in the several variations seen in the species. These variations pose taxonomic problems to all users of Morinda species. Hence a study of variations at phenotypic (morphological) and genotypic levels will be of immense value to the economic exploitation of Morinda citrifolia.

Linnaeus (1753, 1754) circumscribed genus Morinda of Rubiaceae. The family is cosmopolitan, though the species are mostly confined to the warm tropics. The classification of the family and its principal genera are still debated. Presently four subfamilies are recognised (Mabberley, 2008). They are Chinchonoideae, Ixoroideae, Antirheoideae and Rubioideae. Morinda is placed in sub family Rubioideae and in tribe Morindeae. The latter is known for syncarpic fruits and heterogeneous. Subfamily Rubioideae is characterised by the presence of raphides and heterostyly.

Roxburgh (1814) in Hort. Beng. had cited the following species for India, namely M. angustifolia Roxb., M. bracteata Roxb., M. exserta Roxb., M. multiflora Roxb., M. scandens Roxb. and M. tinctoria Roxb. Hooker, J.D. (1880) grouped seven species of Morinda from India in two groups. He placed M. tinctoria, M. angustifolia, M. citrifolia and M. persicaefolia in one group while M. umbellata, M. rigida and M. villosa were in the second group. Gamble (1921) had listed five species for the Madras Presidency namely M. citrifolia, M. angustifolia, M. tinctoria, M. umbellata and M. reticulata.

Razaûmandimbison and Bremer (2011) recently established new circumscription on Morindeae based on a molecular phylogenetic study. The Morindeae, can be diagnosed by their massive T-shaped placentae inserted in the middle of the septum bearing two anatropous ovules per carpel and pyrenes with a single lateral germination slit (Igersheim & Robbrecht 1993). There is WNRF Technical Bulletin : 12 2 frequent occurrence of head-like inûorescences and multiple fruits. Razaûmandimbison et al. (2009) also demonstrated the paraphyly of the type genus Morinda L. with respect to its closely related genera Coelospermum Blume, Gynochthodes Blume, Pogonolobus F. Muell., and Sarcopygme Setch. & Christoph. As a consequence, new generic limits of Morindeae were proposed in order to make Morinda monophyletic and morphologically well-deûned genera were recognized in Morindeae. Hence a total of five genera are now recognized: Appunia, Coelospermum, Gynochthodes, Morinda and Siphonandrium. Razaûmandimbison and Bremer (2011) have effected the nomenclatural changes and outlined taxonomic notes in the tribe Morindeae of Rubiaceae. Some Indian species are placed in the new genus Gynochthodes Blume and others in Morinda L.

Indian species of Morinda that are placed in the new genus Gynochthodes are as follows:

1. Gynochthodes ridsdalei Razafim. & B. Bremer (new name) Synonym: Morinda reticulata Gamble

2. Gynochthodes umbellata (L.) Razafim. & B. Bremer (new name) Synonym : Morinda umbellata L.

3. Gynochthodes villosa (Hook.f.) Razafim. & B. Bremer (new name) Synonym: Morinda villosa Hook.f.

4. Gynochthodes wallichii (Kurz) Razafim. & B. Bremer (new name) Synonym: Morinda wallichii Kurz

Indian species that are retained in Morinda are as follows:

1. Morinda pubescens J.E. Smith (new name) Synonym : Morinda tinctoria Roxb.

2. Morinda angustifolia Roxb.

3. Morinda citrifolia L. WNRF Technical Bulletin : 12 3

Molecular phylogenetic approach has much refined the classification of the family Rubiaceae and its sub categaries especially the tribe Morindeae. The Linnean Morinda was highly paraphyletic but with new circumscription it is monophyletic. A clear picture of M. citrifolia also has emerged and the systematic of M. citrifolia will add new vistas to Noni research in its phytochemical and nutriceutical aspects. It is hoped that phylogenetic relationships of M. citifolia will allow Noni researchers to concentrate on medicinal and biochemical properties of closely related species. 2. State of knowledge

Morinda L. (sensu Razafimandibison and Bremer, 2011)

Small trees or shrubs, rarely lianas. Leaves petiolate (rarely subsessile), chartaceous to coriaceous, glabrous. Inûorescences mostly leaf-opposed (modified terminal inflorescences), sometimes axillary with heads arranged into secondarily compound inflorescences, composed of 1 to 3 heads, sometimes axillary with heads arranged into secondarily compound inflorescences, pedunculate (rarely sessile), branched. Flowers bisexual; corolla tubes much larger than corolla lobes; filaments inserted in corolla throat, linear, glabrous, white; anthers partly exserted (rarely well exserted); disk annular, persistent in fruit; style filiform, terete; stigma bilobate, more or less recurved, not exserted; ovaries inferior, bilocular, with longitudinal false dissepiments, ovaries of all fiowers of a head basally to completely fused; locule biovular. Fruits fused into syncarps. Seed globose or semi-globose.

Gynochthodes Blume (sensu Razafimandibison and Bremer, 2011)

Lianas, rarely shrubs or trees. Leaves petiolate, chartaceous to more or less coriaceous. Inûorescences terminal, terminal on axillary (lateral) shoots, or axillary, solitary or composed of 2 to 10 heads arranged into umbel-like, racemose or cymose or terminal. Flowers polygamous (hermaphrodite and functionally unisexual) or dioecous, mostly completely to basally fused when clustered in heads, sometimes in multiûorous compound dichasia or rarely single, sessile or sometimes pedicel late; corolla tubes shorter than corolla lobes, rarely as long as or longer than corolla lobes; ûlaments inserted in WNRF Technical Bulletin : 12 4 corolla throat, linear, glabrous, white, anthers partly exerted; disk annular, persistent in fruit; styles ûliform, terete; stigmatic lobe bilobate, exserted; ovary inferior, bilocular, with longitudinal false dissepiment separating the ovules, ovaries of all flowers of a head basally to completely fused; locule biovular. Fruits fused into syncarps or simple drupes. Seeds globose or semi-globose.

Taxonomy of Morinda citrifolia varieties

The taxonomic history of Morinda citrifolia varities has been extensively studied by Razafimandimbision et al. (2009). Morinda citrifolia has long been regarded as morphologically heterogeneous (Hooker, 1880; Valeton, 1908; McClatchey, 2003; Nelson &, Elevitch, 2006; Waki et al., 2008); however, there have been no attempts to test its monophyly with a molecular-based phylogeny. Kurz (1877) was the ûrst to establish infraspeciûc subdivisions for M. citrifolia. He included the continental Asian species Morinda bracteata in synonymy of M. citrifolia and recognized two varieties: M. citrifolia var. citrifolia and M. citrifolia var. bracteata. Hooker (1880) described M. citrifolia var. elliptica Hook.f., which was later recognized by Ridley (1918) as a separate species [Morinda elliptica (Hook.f.) Ridley]; this species has since been widely accepted by the Rubiaceae systematists (Puff et al., 2007), consistent with Razaûmandimbison et al. (2009). Today there are three varieties widely recognized in M. citrifolia: var. bracteata, var. citrifolia and var. potteri [= M. citrifolia L. f. potteri (O. Degen.) H. St. John; John, 1984]. Morinda citrifolia var. bracteata is easily distinguished from the other varieties by its conspicuous well-developed bracts subtending the inûorescence and its small fruits. Morinda citrifolia var. potteri differs from the other varieties of M. citrifolia in having variegated leaves and small fruits, foetid when ripe. The large fruited M. citrifolia var. citrifolia appears to have evolved self pollinating ûowers from the ancestral condition of (hermaphroditic) out crossing ûowers (but see also Waki et al., 2008); it flowers and produces fruits year-round.

Description

Morinda angustifolia Roxb., Pl. Corom., 3(2): t. 237. 1815; Fl. India. 2: 201, 1824; Hook. f., Fl. Brit. India 3: 156. 1880; Kanjilal et al., Fl. Assam 3: 79 1939; Deb in Bull. Bot. Surv. India 3: 311. 1961. WNRF Technical Bulletin : 12 5

A large shrub or a small tree, 4-10 m tall. Bark greyish, exfoliating in thin pieces; blaze whitish. Leaves coriaceous, large, elliptic or obovate-lanceolate, (6)15-13(22) × 2-10 cm, varying in size and shape, caudate-acuminate at base, margin undulate, shortly acuminate or acute at apex, glabrous; petiole 3.5 cm; stipules acute. Flowers white, fragrant usually 5-merous ca. 3 cm long, terminal, long on short peduncles, in globose heads, leaf-opposed, peducle elongating in fruit. Calyx nearly truncate or obscurely toothed. Corolla salver- shaped; tube up to 2.5 cm long. Syncarps 2 cm, turbinate, black.

Distribution: Eastern Himalayas, north-east India, Assam, Bihar, Oirssa and Andhra Pradesh up to 1800 m.

Fl. & Fr.: February – October

The root is used for dyeing.

Morinda citrifolia L., Sp. Pl. 176. 1753; DC., Prodr, 4: 466. 1832; Hook.f., Fl. Brit. India 3: 156. 1880; Gamble 2: 652 (459). 1921. Ridsdale in Manilal, Bot. Hist. Malab. 137. 1980; Manilal & Sivar., Fl. Calicut 144. 1982;

Cada-Pilava Rheede, Hort. Malab. 1: 97-98, t. 52. 1678. Morinda bracteata Roxb., Fl. Ind. 2: 198. 1824; Thw., Enum. Pl. Zeyl. 144. 1859. M. citrifolia L. var. bracteata (Roxb.) Hook.f. F. Br. ind. 3: 156. 1880.

Shrub or small tree, 3-6 m tall; trunk not branched, smooth. Branches tetragonal, angles not sharp. Leaves 10-20 × 5-9 cm, elliptic, shining green, glabrous, membranous, 5-6 pairs of distinct lateral nerves, margin entire, apex obtuse or acute-acuminate, base attenuate ending in 1-2 cm long petiole, petiole sinuate; stipules 6-9 × 4-7 mm, rounded, oblong or somewhat semi-circular, glabrous, persistent, often 2-lobed. Inflorescence axillary, usually in every alternate pair of leaves, peduncle c. 1 cm long. Head oblong, 1.5-2.5 × 1-1.5 cm, green, enlarged in fruit, lower flowers of the head open first. Calyx-tube cupular, ca. 2 mm in diameter, lobes truncate or obscure. Corolla white, tube c.12 mm long, cylindrical, glabrous, lobes oblong, 5-6 mm long, recurved, throat hairy, pubescent. Stamens yellowish, somewhat exserted. Heads of fruit ovoid, glossy, 4-6 × 3-4 cm, whitish or pale-yellow, pyreness compressed on WNRF Technical Bulletin : 12 6 one side, winged on edges, ca. 8 × 5 mm, with basifixed white hairs specially on edges and wing.

Cada-Pilava (M. citrifolia) finds a place in Hortus Malabaricus of van Rheede (1678). The details outlined by him, enshrine the indigenous pre- Ayurvedic knowledge of the hereditary medical practitioners of the Kerala state.

Distribution: Warmer parts of India, Sri Lanka, Bangladesh, Malaya, N. Australia and Pacific Islands, cultivated elsewhere. In Pakistan it is cultivated as an ornamental.

Fl. & Fr.: Almost throughout the year.

Its powers: The juice pressed out from the whole plant and cooked with oil from the leaves is good for parts affected by gout, for which also is helpful smearing of the root crushed and mixed with water. Cultivated widely in many places throughout India, found also as an escape, but not truly wild. The roots furnish a valuable red eye.

Morinda pubescens J.E. Smith in Rees. Cyclop. 24: n. 3. 1813.

Leaves narrowly linear-oblong var. stenophylla

Leaves broadly obovate-elliptic var. pubescens var. pubescens: Verdc. in Kew Bull. 37: 543. 1983. M. tinctoria Roxb. Fl. ind. 2: 197. 1824, non Noronha 1790; Hook.f., Fl. Brit. India 3: 156. 1880; Gamble 2: 652 (459). 1921. Morinda coreia Buch-Ham., Trans. Linn. Soc. London 13: 537. 1822; M. tinctoria Roxb. var. tomentosa (Heyne ex Roth) Hook. f. Fl. Brit. India 3: 156. 1880; M. tomentosa Heyne ex Roth, Nov. Pl. Sp. 147. 1821.

Small tree to 6(-10) m, bark thick, rough, fissured and somewhat corky. Branchlets often with a characteristic thin pallid to yellowish bark. Leaves (broadly-) elliptic, less frequently linear-lanceolate or obovate, 6-12(-20) × (1.5-) 3.5-6(-8) cm, above glabrous, below glabrous or tomentose, apex acute to acuminate, base to cuneate, frequently decurrent, lateral nerves 4-9 pairs, domatia in axils usually hairy, rarely glabrous. Stipules deltoid to narrowly WNRF Technical Bulletin : 12 7 triangular, 3-8 mm long, apex acute, sometimes slightly bifid. Petiole up to 3.5 cm. Inflorescence a solitary, axillary or rarely terminal, ovoid or globose, capitulum, often appearing leaf-opposed or subtended by a reduced leaf, pedicels up to 3.5 cm. Flowers 5(rarely 6)-merous. Calyx limb short, truncate. Corolla white, tube 10-15 mm, inside glabrous in the throat, lobes oblong, 6- 8 mm long, recurved. Stamens exserted or partially included, anthers oblong, 4-5 mm. Style 10-18 mm, stigma 2-lobed. Syncarp 1.5-2.5 cm diam.

Distribution: Sri Lanka, India, Malay Archipelago.

Fl. & Fr.: May – Nov. var. stenophylla (Spreng.) Kumari in Henry et al. Flora of Tamilnadu 2: 14. 1987.M. stenophylla Spreng. Syst. Veg. 1: 749. 1824. M. tinctoria Roxb. var. stenophylla (Spreng.) Gamble, Fl. Pres. Madras 652 (460). 1921.

A shrub, up to 3 m tall, stem pubescent. Leaves narrowly linear-lanceolate or oblong, 6-12.5 × 1.2-1.8 cm, cuneate at base, entire margin, acute-acuminate at apex, slightly pubescent; petiole very short, 0.6 cm; stipules very large, linear, 1.5 × 0.5 cm. Flowers in axillary cyme, white. Syncarp 0.5 × 0.4 cm.

Distribution: Andhra Pradesh and Tamil Nadu.

Fl. & Fr.: May – Nov.

Gynochthodes ridsdalei Razafim. and B. Bremer ADANSONIA, sér. 3, 2011, 33 (2) Morinda reticulata Gamble), Gamble in Bull. Misc. Inform. 248. 1920 & in Fl. Pres. Madras 652 (460) 1921; A.N. Henry et al. in J. Bomaby Nat. Hist. Soc. 75(3): 690. 1978; A.N. Henry & Swamin. in Indian J. For. 3(2): 140. 1980; Vajravelu & P. Daniel in Jain & Sastry. Mat. Cat. Threat. Pl. India 24. 1983; Swamin. in A.N. Henry et al., Fl. Tamil Nadu Ser. I. Analy. 2: 14 . 1987.

Climbing shrubs, stem terete, slender. Leaves opposite, simple; lamina elliptic to oblanceolate, 2.5-17 × 0.5-5 cm, coriaceous, shining, cuneate to attenuate at base, entire, acuminate (acumen up to 2.5 cm long) at apex; nerves 5-12 pairs, midrib prominent, raised beneath, less prominent and sunken above; lateral nerves prominent beneath less above, curved upwards, joined and looped towards the margin, reticulation fine. stipules small, connate at base; WNRF Technical Bulletin : 12 8 petioles upto 12 mm long, grooved adaxially, rounded abaxially, turgid at base; Inflorescence terminal, compound umbels; peduncles 10-25 mm long, glabrescent; bracts small, cupular, glabrescent. Flowers sessile or subsessile, bisexual; calyx-tube saucer-shaped, ca. 1 mm long, glabrous, lobes 0, rim wavy, glabrous. Corolla tube short, up to 1 mm long; lobes 4, linear-oblong, ca. 3 × 1.5 mm, thick, cottony wooly within, glabrous without, entire, obtuse and mucronate at apex, incurved. Stamens 4, alternipetalous; filament short ca. 1 mm long, glabrous; anthers oblong glabrous; anthers oblong-linear, c. 2 mm long, 2 loculed, introrse, dorsifixed. Ovary semi-inferior, glabrous, small, globose, c. 0.5 × 0.5 mm. Fruits globose, 9-18 × 9-18 mm, syncarpous, pyrenes pyriform. Seeds oblong-obovoid, not winged.

Distribution: A very rare climber in evergreen forests. Southern Western Ghats. 200-850 m. Tamil Nadu (Kanniyakumari district) and Kerala (Thiruvananthapuram district) Endemic.

Fl. & Fr.: June- October.

Gynochthodes umbellata (L.) Razafim. and B. Bremer ADANSONIA, sér. 3 • 2011 • 33 (2) (Morinda umbellata L.) Sp. Pl. 176. 1753; Hook. f., Fl. Brit ind. 3: 157 1880; Gamble 2: 652 (460) 1921) Ridsdale in Manilal, Bot Hist. Hort Malab. 137. 1980. – Morinda pada-vara Juss. ex Schult. in Roem. & Schult., Syst. Veg. 5: 216. 1819. Morinda tetrandra Jack, Malayan Misc. 1(5): 13. 1820. nom. illeg. (incl. type of M. pada-vara Schult. 1819).

Pada vara Rheede, Hort. Malab. 7: 51-52, t. 27. 1688.

A climbing or straggling shrub, up to 3 m tall. Leaves coriaceous, shining, broadly obovate-elliptic or linear-lanceolate, 6-17.5 × 2-9 cm, cuneate- acuminate at base, margin entire, acute at apex, glabrous or pubescent above, tomentose below, nerves very distinct; petiole 3 cm; stipule connate, acuminate. Flowers in terminal umbellate heads, white, peduncle pubescent. Calyx c. 1 mm long, truncate. Corolla tube c. 1 mm long. villous within, lobes 4, recurved. Stamens 4. Syncarp 1.2 cm wide, occasionally separate, irregularly lobed 0.5 × 4 cm, green; pyrenes pyriform. Seeds oblong.

Distribution: Sri Lanka, India, through to China, Japan, N. Australia WNRF Technical Bulletin : 12 9

Fl. & Fr.: January to September.

The name Padavara (Malayalam script on t. 27) is still used, meaning of which is not clear. It is also called Kudalchurukki; kudal means intestine and churukki means to shrink, referring to its use as medicine for intestinal disorders. It occurs in sacred groves through out Malabar.

Gynochthodes villosa (Hook.f.) Razafim. and B.Bremer ADANSONIA, sér. 3 2011 33 (2) Morinda villosa Hook. f., Fl. Brit. India 3: 158. 1880; Kanjilal et al., Fl. Assam 3: 80 1939.

A Climbing shrub; branches more or less hirsute, longitudinally ribbed. Leaves

2-10 × 2.5-3.5 cm, elliptic to elliptic-lanceolate, pubescent above, hirsute beneath, pale greenish when dry; lateral nerves prominent, 10-15 on either half; base narrowed into the petiole. Flowers usually 4-5-merous in terminal, long pedunculate umbels. Corolla white, tube short. Syncarp deep orange, 1.5 cm across.

Distribution: Khasi Hills Cachar, Kameng, Tirap; 500-800 m, in primary forests.

Fl. & Fr.: April - October 3. Objectives z Collections of accessions of elite plants of Morinda citrifolia with variations from varied climatic-eco regions submit processed herbarium specimens to WNRF. z Study of morphological variations, floral biology and fruit setting z Identification of elite varieties with higher yield of fruits and with maximum size. z Study biological properties and analyse Phytochemical variations in various accessions by means of GC-MS (outsourcing). z Correlation of field data of superior varieties with a thorough study of genetic variations by applying molecular markers. WNRF Technical Bulletin : 12 10

4. Experimental details

Citation of specimens: The following species citation with their accession number is given here separately for collections made in states of Tamilnadu and Kerala. The RHT number refers to individual collected samples for each species from various district of the above states.

The distributional status shows the following features. M. citrifolia as a natural population is totally absent in Tamilnadu state except in cultivation by Farmers and Gardeners. Within Kerala state it is dominant in the middle and northern regions. The population are very robust along coastal, back water and riverine areas of the state. In Kasaragod district the coastal populations are extra ordinarily dominant. Undisturbed coastal areas harbor several isolated populations. Islands along coastal region and sand dunes along rivers also make the populations flourish. The hills of the Western Ghats in Kerala do not have significant of presence of M. citrifolia. In the plains of Kerala there is the dominance of M. pubescens with several Morphoforms. However the linear leafed form of M. Pubescens var. stenophylla is rather rare. The table below in indicates the details of field days and collection work. Data on field trips Field trips Year Months No. of days No. of collections 2 2009 May 4 11 2 June 4 27 1 August 1 18 2 Sepetember 2 20 2 October 4 26 3 November 6 71 1 December 3 28 13 24 201 WNRF Technical Bulletin : 12 11

Field trips Year Months No. of days No. of collections 1 2010 February 4 53 5 March 5 100 1 April 1 1 4 May 4 43 1 June 1 15 2 July 2 27 2 August 2 18 7 September 6 90 1 October 2 31 4 November 5 59 2 December 2 24 30 34 461 Field trips Year Months No. of days No. of collections 2 2011 January 2 42 2 February 2 27 2 March 5 35 1 April 1 8 1 May 2 21 1 June 2 36 2 July 2 108 1 August 1 20 1 September 1 34 13 18 331 WNRF Technical Bulletin : 12 12

Field trips Year Months No. of days No. of collections 2 2012 January 3 24 2 February 4 84 4 7 108 Trips No of days No. of collections 60 83 1101 TAMILNADU

Morinda citrifolia var. bracteata

Kannyakumari — RHT: 56758, 56759, 56760, 56838, 56839, 56840, 56841, 56842, 56843, 56844, 56845, 56846, 56847, 56848, 56849, 56850, 56851, 56852, 56853; Tiruchirappalli – RHT: 56006, 56597, 56598, 56599, 56600, 56601, 56602, 56603, 56604, 56605, 56606, 56607.

Morinda pubescens var. pubescens

Dindigul – RHT: 56037, 56038 (Seeds); Kannyakumari – RHT: 56761, 56762, 56763, 56764, 56765, 56766, 56767, 56768, 56769; Madurai – RHT: 56011; Nagapattinam — RHT: 56955, 56956, 56961, 56963, 56964; Pasumpon – RHT: 56689, 56783, 56937, 56938; Perambalur – RHT: 56012, 56013; Pudukottai – RHT: 56787, 56788, 56789, 56790, 56791, 56792, 56793; Ramanathapuram – RHT: 56008, 56009, 56221, 56222, 56223, 56696, 56697, 56698, 56700, 56927, 56928, 56929, 56930, 56933, 56934; Sivagangai – RHT: 56010, 56691, 56692, 56785, 56940, 56941, Thanjavur – RHT: 56218, 56220, 56786, 56919, 56920, 56921, 56922, 56923, 56924, 56925, 56926, 56948, 56949, 56950, 56952, 56953, 56954, 56958; Thiruvallur – RHT: 56024, 56025; Thiruvarur – RHT: 56215, 56217, 56711, 56908, 56909, 56914, 56915, 56916, 56917, 56918, 56944, 56951, 56957; Tiruchirappalli – RHT: 56007, 56014, 56015, 56021, 56708, 56709, 56710, 56866, 56900, 56901, 56902, 56903, 56904, 56905, 56906, 56907; Tirunelveli – RHT: 56399, 56400, 56401, 56402, 56403, 56404; Virudhunagar – RHT: 56393, 56394, 56395, 56396, 56397, 56398, 56687, 56935, 56936. WNRF Technical Bulletin : 12 13

Morinda pubescens var. stenophylla

Kanchipuram – RHT: 56027; Kannyakumari – RHT: 56701, 56702, 56703, 56704, 56705, 56706, 56707; Madurai – RHT: 56680, 56681, 56682, 56683, 56684; Nagapattinam – RHT: 56962; Pasumpon – RHT: 56688, 56690, 56939; Ramanathapuram – RHT: 56699, 56784, 56931, 56932, 56946; Sivagangai RHT: 56693, 56694, 56942, 56943; Thanjavur – RHT: 56219, 56695, 56945, 56947, 56959, 56960; Thiruvallur – RHT: 56026, 56867, 56868; Thiruvarur – RHT: 56216; Tiruchirappalli – RHT: 56028, 56029, 56030; Tirunelveli – RHT: 56405, 56406, 56407; Virudhunagar – RHT: 56493, 56685, 56686.

Gynochthodes ridsdalei (Morinda reticulata)

Thiruvarur – RHT: 56712, 56910, 56911, 56912, 56913.

Gynochthodes umbellata (Morinda umbellata)

Thiruvallur – RHT: 56016, 56017, 56018, 56019, 56020, 56022, 56023.

KERALA

Morinda angustifolia

Ernakulam – RHT: 56322, 56323, 56579, 56580, 56581, 56582, 56584, 56585, 56586, 56587, 56589, 56590, 56591, 56620, 56621, 56622, 56623; Thiruvananthapuram – RHT: 56596, 56609, 56613, 56614, 56615, 56616, 56617, 56618, 56619, 56743, 56744, 56745, 56746, 56747, 56748, 56772, 56773, 56776, 56777, 56778, 56779, 56780, 56781, 56858, 56860, 56861, 56862, 56863, 56864, 56972, 56974, 56975, 56976, 56977, 57019, 57020, 57021; Thrissur – RHT: 56191, 56193.

Morinda citrifolia var. bracteata

Allpuzha – RHT: 56494, 56495, 56500, 56501, 57129, 57133, 57134; Calicut – RHT: 56004, 56276, 56277, 56278, 56279, 56280, 56281, 56305, 56306, 56307, 56308, 56309, 56310, 56311, 56337, 56338, 56339; Ernakulam – RHT: 56041, 56042, 56043, 56044, 56045, 56046, 56208, 56209, 56210, WNRF Technical Bulletin : 12 14

56211, 56212, 56213, 56224, 56225, 56226, 56227, 56228, 56229, 56230, 56231, 56232, 56233, 56347, 56348, 56349, 56350, 56351, 56352, 56353, 56354, 56355, 56356, 56357, 56358, 56359, 56360, 56361, 56362, 56363, 56364, 56365, 56366, 56367, 56368, 56369, 56370, 56371, 56372, 56373, 56374, 56375, 56376, 56377, 56378, 56379, 56448, 56449, 56450, 56451, 56452, 56453, 56454, 56455, 56456, 56457, 56508, 56509, 56510, 56511, 56512, 56513, 56514, 56515, 56516, 56517, 56518, 56519, 56520, 56521, 56522, 56523, 56524, 56554, 56555, 56556, 56557, 56558, 56559, 56560, 56561, 56562, 56563, 56583, 56588, 56592, 56593, 56822, 56823, 56824, 56825, 56826, 56827, 56828, 56829, 56830, 56831, 56832, 56833, 56834, 56835, 56876, 56877, 56878, 56879, 56880, 56881, 56882, 56883, 56884, 56885, 56886, 56887, 56888, 56889, 56890, 57135, 57136, 57137, 57138, 57139, 57140, 57141, 57142, 57143, 57144, 57145, 57146, 57147, 57148, 57149, 57150, 57151, 57152, 57153, 57154, 57155, 57156, 57157, 57158, 57159, 57160, 57161, 57162, 57163, 57164, 57165, 57166, 57167, 57168, 57169, 57170, 57171, 57172, 57173, 57174, 57175, 57176, 57177, 57178, 57179, 57180, 57181, 57182, 57183, 57184, 57185, 57186, 57187, 57188, 57189, 57190, 57191, 57192, 57193, 57194, 57195, 57196, 57197, 57198, 57199, 57200, 57201, 57202, 57203, 57204, 57205, 57206, 57207, 57208, 57209, 57210, 57211, 57212, 57213, 57214, 57215, 57216, 57217, 57218, 57219, 57220, 57221, 57222, 57223, 57224, 57225, 57226, 57227, 57228, 57229, 57230, 57231, 57232, 57233, 57234; Kannur – RHT: 56636, 56637, 56638, 56639, 56640, 56641, 56642, 56643, 56644, 56645, 56646, 56647, 56648, 56649, 56650; Kasaragod – RHT: 56003, 56005, 56340, 56341, 56342, 56343, 56344, 56345, 56346, 56654, 56655, 56656, 56658, 56668, 56672, 56675, 56676, 56677, 56678, 56679; Kollam – RHT: 56713, 56714, 56715, 56716, 56717, 56718, 56719, 56720, 56721, 56722, 56723, 56724, 56725, 56726, 56727, 56728, 56729, 56730, 56731, 56732, 56733, 56734, 56735, 56736, 56737, 56738, 56739, 56740, 56741; Kottayam – RHT: 56245, 56246, 56247, 56248, 56262, 56263, 56264, 56265, 56266, 56267, 56268, 56269, 56270, 56271, 56272, 56273, 56274, 56275; Kozhikode – RHT: 56312, 56313, 56314, 56315, 56316, 56317, 56318, 56319, 56320, 56321, 56382, 56386, 56391, 56564, 56652, 56742; Mahe — RHT: 56565, 56566, 56567, 56568, 56569, 56570, 56571, 56572, 56573, 56574, 56575, 56576, 56577, 56578, 56624, 56625, 56626, 56627, 56628, 56629, 56630, 56631, WNRF Technical Bulletin : 12 15

56632, 56633, 56634, 56635; Thiruvananthapuram – RHT: 56503, 56504, 56505, 56506, 56507, 56540, 56541, 56542, 56543, 56544, 56545, 56546, 56547, 56548, 56549, 56749, 56750, 56751, 56752, 56753, 56754, 56755, 56756, 56757, 56782, 56794, 56795, 56796, 56797, 56798, 56799, 56800, 56801, 56802, 56803, 56804, 56805, 56806, 56807, 56808, 56809, 56810, 56811, 56812, 56813, 56814, 56815, 56816, 56817, 56818, 56819, 56820, 56821, 56854, 56855, 56856, 56857, 57053, 56859, 56970, 56971, 56973, [56031, 56032, 56033, 56034, 56035 (Seeds)], 56978, 56979, 56980, 56981, 56982, 56983, 56984, 56985, 56986, 56987, 56988, 56989, 56990, 56991, 56992, 56993, 56994, 56995, 56996, 56997, 56998, 56999, 57000, 57001, 57002, 57003, 57004, 57005, 57006, 57007, 57008, 57009, 57010, 57011, 57012, 57013, 57014, 57015, 57016, 57017, 57018, 57022, 57023, 57024, 57025, 57026, 57027, 57028, 57029, 57030, 57031, 57032, 57033, 57034, 57035, 57036, 57037, 57038, 57039, 57040, 57041, 57042, 57043, 57044, 57045, 57046, 57047, 57048, 57049, 57050, 57051, 57052, 57054, 57055, 57056, 57057, 57058, 57059, 57060, 57061, 57062, 57063, 57064, 57065, 57066, 57067, 57068, 57069, 57070, 57071, 57072, 57073, 57074, 57075, 57076, 57077, 57078, 57079, 57080, 57081, 57082, 57083, 57084, 57085, 57086, 57087, 57088, 57089, 57090, 57091, 57092, 57093, 57094, 57095, 57096, 57097, 57098, 57099, 57100, 57101, 57102, 57103, 57104, 57105, 57106, 57107, 57108, 57109, 57110, 57111, 57112, 57113, 57114, 57115, 57116, 57117, 57118, 57119, 57120, 57121, 57122, 57123, 57124, 57125, 57126; Thrissur – RHT: 56039, 56040, 56047, 56048, 56049, 56050, 56051, 56052, 56055, 56056, 56189, 56190, 56197, 56200, 56201, 56202, 56214, 56282, 56283, 56284, 56285, 56286, 56287, 56288, 56289, 56290, 56291, 56293, 56294, 56295, 56296, 56297, 56298, 56299, 56300, 56301, 56302, 56303, 56304, 56408, 56409, 56410, 56411, 56412, 56415, 56416, 56417, 56418, 56419, 56420, 56421, 56424, 56427, 56431, 56432, 56433, 56434, 56435, 56436, 56437, 56438, 56441, 56442, 56443, 56444, 56445, 56446, 56447, 56463, 56464, 56465, 56466, 56467, 56468, 56469, 56470, 56471, 56472, 56473, 56474, 56475, 56476, 56477, 56478, 56479, 56480, 56481, 56482, 56483, 56484, 56485, 56486, 56487, 56488, 56490, 56491, 56492, 56525, 56526, 56527, 56528, 56529, 56530, 56531, 56532, 56533, 56534, 56535, 56536, 56553, 56869, 56870, 56871, 56872, 56873, 56874, 56875, 56891, 56892, 56894, 56895, 56896, 56897, 56898, 56899. WNRF Technical Bulletin : 12 16

Morinda citrifolia var. citrifolia

Allpuzha – RHT: 56496, 56497, 56498, 56499, 56502, 57127, 57128, 57130, 57131, 57132; Kasaragod – RHT: 56657, 56659, 56660, 56661, 56662, 56663, 56664, 56665, 56666, 56667, 56669, 56670, 56671, 56673, 56674; Kozhikode – RHT: 56324, 56325, 56326, 56327, 56328, 56329, 56330, 56331, 56332, 56333, 56334, 56335, 56336, 56380, 56381, 56383, 56384, 56385, 56387, 56388, 56389, 56390, 56392, 56552, 56651, 56653; Thrissur – RHT: 56001, 56002, 56053, 56054, 56188, 56194, 56195, 56196, 56198, 56199, 56203, 56204, 56205, 56206, 56207, 56234, 56235, 56236, 56237, 56238, 56239, 56240, 56241, 56242, 56243, 56244, 56249, 56250, 56251, 56252, 56253, 56254, 56255, 56256, 56257, 56258, 56259, 56260, 56261, 56292, 56413, 56414, 56422, 56423, 56425, 56426, 56428, 56429, 56430, 56439, 56440, 56458, 56459, 56460, 56461, 56462, 56489, 56865, 56893.

Morinda pubescens var. pubescens

Thiruvananthapuram – RHT: 56537, 56538, 56539, 56550, 56551, 56594, 56595, 56608, 56610, 56611, 56612, 56770, 56771, 56774, 56775, 56965, 56966, 56967, 56968, 56969, 56036 (Seed); Thrissur – RHT: 56192. WNRF Technical Bulletin : 12 17 Documentation of field work and number herbarium collections WNRF Technical Bulletin : 12 18

Morinda angustifolia Roxb. : habit

Gynochthodes umberllata (L.,) Razafim, and B. Bremer (Morinda umbellata L.) : 1. habit; 2 & 3 flower WNRF Technical Bulletin : 12 19

Gymochthodes ridsdalei Razafim. and B. Bremer (Morinda reticulata Gamble) : 1. habit; 2. flower, 3 & 4. calyx; 5. corolla split open; 6. stamen

Morinda pubescens J. E. Smith var. pubescens : 1. habit; 2. flower; 3. corolla splitopen; 4. stamen; 5. pistil, 6. syncarp. WNRF Technical Bulletin : 12 20

Morinda citrifolia L. : 1 & 10. habit; 2. inflorescence; 3. flower, 4 & 5 corolia, split open; 6. stamen; 7. pistil; 8 & 9. ovary, t.s. & I.s.; 11. syncarp, 12. seed 5. Experimental findings i Antimicrobial analysis MIC and MBC determinations in M. citrifolia MIC test of Noni fruit was done selectively against eight organisms that were found susceptible in agar diffusion assay. They are Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, Salmonella paratyphi, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus and Staphylococcus faecalis. MIC values also showed that extracts were able to inhibit both gram positive bacteria and gram negative bacteria at lower concentrations, The lowest MIC for Morinda citrifolia fruit ethanol extract was recorded against S. paratyphi, (0.03mg/ ml), Vibrio cholerae (0.03mg/ml), P. aeruginosa (0.06mg/ml), E. coli (0.12mg/ml), S. aureus (0.25mg/ml), B. cereus (0.25mg/ml) and S. faecalis (0.5mg/ml) as detailed in (Table-1). The ethanol extract gave an MBC value for S. paratyphi (0.12mg/ml), Vibrio cholera (0.12mg/ml), P. aeruginosa (0.12mg/ml), and E. coli ((0.12mg/ml) respectively, while for and B. subtilis, B. cereus S. faecalis and S. aureus the MBC values were 0.5-1 mg/ml, WNRF Technical Bulletin : 12 21 respectively (Table-2). Petroleum ether extract gave an MBC value for S. paratyphi (0.5mg/ml), Vibrio cholerae (0.5mg/ml), P. aeruginosa (0.5mg/ ml), and E. coli ((1mg/ml) respectively, while for and B. subtilis, B. cereus S. faecalis and S. aureus the MBC values were 1-4 mg/ml, respectively. For benzene, dichloromethane and aqueous extract, the MBC values were resistant (R) for all the pathogens. Agents with low activity against a particular organism usually gives high MIC and MBC values, while a highly reactive agent gives low MIC and MBC values. The MIC and MBC techniques are used to evaluate the efficacies of antimicrobial agents and in this study, the MIC and MBC values tend to support the results obtained in the antibacterial screening by agar diffusion showing clearly that the ethanol extracts were more potent than either petroleum ether, benzene, dichloromethane, and aqueous extracts.

Minimum Inhibitory Concentration (MIC) of fruits of ethanol extract in Morinda citrifolia L. WNRF Technical Bulletin : 12 22

Table 1 :Minimum Inhibitory Concentration (MIC) of fruits in Morinda citrifolia L.

MIC values < 0.5 mg/ml (500 µg /ml) were considered to be active, Antibiotic –C1- Kanamycin 30 mg/ml, C2- Gentamycin 10mg/ml.

Table 2 : Minimum bactericidal concentration of fruits in Morinda citrifolia L.

Resistance (R) MBC values < 0.5 mg/ml (500 µg /ml) were considered to be active WNRF Technical Bulletin : 12 23 ii Phytochemistry GC-MS Analysis GC-MC plays a key role in the analysis of unknown components of plant origin. GC-MS ionizes compound and measures their mass numbers. Ionization method includes EI (Electron Ionization). The EI method produces ions by colliding thermal electrons emitted from a filament with sample gas molecules. This method provides high stability in ionization and obtained mass spectra show good reproducibility. WNRF Technical Bulletin : 12 24 iii Phytochemistry - HPLC HPLC analysis was carried out for the component separated in thin layer chromatography. It was performed on Schimedzu, Spintrom HPLC – 530.

A C18 gravity column (Aldrich Cat No - 37,763-5, 10 g) was wetted with 100 ml of HPLC grade methanol and then washed with 50 ml of Milli-Q water. A 50 ml aliquot of crude extract was added to the column and allowed to adsorb. The column was washed with 100 ml of Milli-Q water to remove water soluble compounds, and then with 50 ml of 30% methanol to remove less polar material. A 70% methanol solution was then added to the column and 2.0 ml fractions collected.

Fractions were analysed on a Spherisorb ODS-2 C18 analytical column (250 x 4.6 mm, 5 micron) using a 20 minute linear gradient of 15-35% acetonitrile in 8 mM ammonium acetate, with a 10 minute isocratic elution at 35% acetonitrile (8 mM ammonium acetate). Peak detection was achieved using a GBC LC 1250 fast scanning UV/Vis detector set at 238 nm. Eluting peaks were scanned between 200 nm and 300 nm with 1 nm intervals to determine absorbance maxima and minima. Free Sample

AMPHIGENE RESEARCH LABORATORIES # 392 / 2, Mariamman Kovil, Nagai Road, Thanjavur –613 501. Phone: 03462 267 277 / E-mail: [email protected] WNRF Technical Bulletin : 12 25

iv Antioxidant activity

The ethanolic extracts of fruits and leaves of M. citrifolia were assessed for their free radical scavenging activities against superoxide radical, hydroxyl radical, DPPH radical and inhibition of lipid peroxidation. Both extracts showed significant antioxidant activity in vitro at concentration ranging from 10-100µg/ ml against free radical, superoxide radical and hydroxyl radical. IC50 value represents the concentration of test extract where the inhibition of test activity reached 50% and it was calculated by linear regression analysis. WNRF Technical Bulletin : 12 26

Antioxidant activity of Ripened fruit ethanol extract of Morinda citrifolia.

DPPH Radical scavenging Assay

Concentrationµg/mL O.D Mean % of inhibition

Control 1.029 0.975 — 0.920 10 0.822 0.806 17% 0.790 20 0.760 0.744 23% 0.728 30 0.534 0.518 46% 0.503 40 0.460 0.442 54% 0.424 50 0.370 0.347 64% 0.323 100 0.308 0.280 71% 0.338

Antioxidant activity of Leaf ethanol extract of Morinda citrifolia

DPPH Radical scavenging Assay

Concentrationµg/mL O.D Mean % of inhibition

Control 0.536 0.521 — 0.507 10 0.425 0.421 19% 0.418 20 0.314 0.321 38% 0.328 WNRF Technical Bulletin : 12 27

30 0.220 0.226 56% 0.231 40 0.175 0.164 68% 0.153 50 0.121 0.132 74% 0.142 100 0.089 0.088 83% 0.088

Antioxidant activity of Ripened fruit ethanol extract of Morinda citrifolia.

Lipid peroxidation

Concentrationµg/mL O.D Mean % of inhibition

Control 0.378 0.367 — 0.356 10 0.207 0.202 44% 0.198 20 0.145 0.130 64% 0.116 30 0.105 0.106 71% 0.107 40 0.098 0.100 72% 0.102 50 0.153 0.146 60% 0.140 100 0.221 0.197 46% 0.174 WNRF Technical Bulletin : 12 28

Antioxidant activity of Ripened fruit ethanol extract of Morinda citrifolia.

Hydroxyl radical scavenging activity

Concentrationµg/mL O.D Mean % of inhibition

Control 0.972 0.967 — 0.963 10 0.620 0.606 37% 0.592 20 0.539 0.540 44% 0.542 30 0.484 0.474 50% 0.464 40 0.383 0.391 59% 0.399 50 0.366 0.355 63% 0.344 100 0.330 0.340 64%

Antioxidant activity of Leaf ethanol extract of Morinda citrifolia.

Hydroxyl radical scavenging activity

Concentrationµg/mL O.D Mean % of inhibition

Control 0.893 0.878 — 0.864 10 0.424 0.433 50% 0.443 20 0.344 0.355 59% 0.366 WNRF Technical Bulletin : 12 29

30 0.308 0.315 64% 0.321 40 0.290 0.282 67% 0.273 50 0.256 0.248 71% 0.241 100 0.211 0.206 76%

Antioxidant activity of Ripened fruit ethanol extract of Morinda citrifolia.

Superoxide radical assay

Concentration OD OD After- Mean % of µg/mL before inhibition

Control 0.092 1.037 0.945 1.061 — 0.021 1.197 1.176 10 0.193 1.111 0.918 0.937 11% 0.197 1.153 0.956 20 0.361 1.173 0.812 0.856 19% 0.269 1.169 0.900 30 0.363 1.091 0.728 0.692 34% 0.351 1.007 0.656 40 0.376 1.104 0.728 0.638 39% 0.449 0.998 0.549 50 0.513 0.859 0.346 0.399 62% 0.604 1.056 0.452 100 1.077 1.898 0.821 0.794 25% WNRF Technical Bulletin : 12 30

Antioxidant activity of Leaf ethanol extract of Morinda citrifolia

Superoxide radical assay

Concentration OD OD After- Mean % of µg/mL before inhibition

Control 0.019 1.022 1.003 1.002 — 0.017 1.018 1.001 10 0.020 0.961 0.947 0.949 5% 0.028 0.980 0.952 20 0.038 0.953 0.915 0.921 8% 0.054 0.981 0.927 30 0.057 0.961 0.904 0.901 10% 0.062 0.960 0.898 40 0.089 0.956 0.867 0.865 13% 0.080 0.943 0.863 50 0.090 0.720 0.630 0.613 38% 0.098 0.693 0.595 100 0.197 0.567 0.370 0.389 61% 0.177 0.585 0.408 WNRF Technical Bulletin : 12 31 v. Molecular Systematics

Collection from varied climatic eco regions WNRF Technical Bulletin : 12 32

Electrophoresis The PCR products (14 µl) were mixed with 6× gel loading buffer (2 µl) and loaded onto an agarose (1.5% w/v) gel electrophoresis in 0.5× TBE (Tris- Borate- EDTA) buffer at 100 V for 150 min. The gel was stained in ethidium bromide solution, visualized under UV in GEL DOC (UK) Image Analysis System with UViTEC analysis package (Cambridge, UK). List of Primers used

S. No. RAPD PRIMER Sequence Description 1 S1 GTTTCGCTCC 2 S2 TGATCCCTGG 3 S3 CATCCCCCTG 4 S4 GGACTGGAGT 5 S5 TGCGCCCTTC 6 S6 TGCTCTGCCC 7 S7 GGTGACGCAG 8 S8 GTCCACACGG 9 S9 TGGGGGACTC 10 S10 CTGCTGGGAC 11 S11 GTAGACCCGT 12 S12 CCTTGACGCA 13 S13 TTCCCCCGCT 14 S14 TCCGCTCTGC 15 S15 GGAGGGTGTT 16 S16 TTTGCCCGGA 17 S17 AGGGAACGAG 18 S18 CCACAGCAGT 19 S19 ACCCCCGAAG 20 S20 GGACCCTTAC 21 S21 CAGGCCCTTC contd... WNRF Technical Bulletin : 12 33

S. No. RAPD PRIMER Sequence Description 22 S22 TGCCGAGCTG 23 S23 AGTCAGCCAC 24 S24 AATCGGGCTG 25 S25 AGGGGTCTTG 26 S26 GGTCCCTGAC 27 S27 GAAACGGGTG 28 S28 GTGACGTAGG 29 S29 GGGTAACGCC 30 S30 GTGATCGCAG 31 S31 CAATCGCCGT 32 S32 TCGGCGATAG 33 S33 CAGCACCCAC 34 S34 TCTGTGCTGG 35 S35 TTCCGAACCC 36 S36 AGCCAGCGAA 37 S37 GACCGCTTGT 38 S38 AGGTGACCGT 39 S39 CAAACGTCGG 40 S40 GTTGCGATCC 41 S41 ACCGCGAAGG 42 S42 GGACCCAACC 43 S43 GTCGCCGTCA 44 S44 TCTGGTGAGG 45 S45 TGAGCGGACA 46 S46 ACCTGAACGG 47 S47 TTGGCACGGG 48 S48 GTGTGCCCCA 49 S49 CTCTGGAGAC contd... WNRF Technical Bulletin : 12 34

S. No. RAPD PRIMER Sequence Description 50 S50 GGTCTACACC 51 S51 AGCGCCATTG 52 S52 CACCGTATCC 53 S53 GGGGTGACGA 54 S54 CTTCCCCAAG 55 S55 CATCCGTGCT 56 S56 AGGGCGTAAG 57 S57 TTTCCCACGG 58 S58 GAGAGCCAAC 59 S59 CTGGGGACTT 60 S60 ACCCGCTCAC

Preliminary screening for RAPD-PCR primers From a preliminary, screen of 60 RAPD primers from Bio Basic Inc., 23 primers were selected based on reproducibility of amplifications. S1- S20 series : S1, S5, S6, S8, S11, S14, S17. S21 - S40 series: S22, S23, S29, S30, S33, S34, S38. S41-S60 series: S41, S42, S43, S45, S47, S48, S51, S58, S60. WNRF Technical Bulletin : 12 35

RAPD Gel Images WNRF Technical Bulletin : 12 36 WNRF Technical Bulletin : 12 37 WNRF Technical Bulletin : 12 38

RAPD markers produced by selected primers

S.No Primer Total Range of size bands % of no. bands of fragments polymorphic polymorphism ( Kb) 1 S 1 6 0.28 - 0.85 4 66.6 2 S 5 1 0.65 0 0 3 S 6 1 0.66 0 0 4 S 14 5 0.97 -1.75 5 100 5 S 17 5 0.49 - 0.83 4 80 6 S 22 1 0.97 0 0 7 S 23 6 0.2- 0.90 6 100 8 S 29 6 0.2- 0.96 6 100 9 S 30 6 0.54 - 1.0 6 100 10 S 38 6 0.3 - 1.40 6 100 11 S 41 10 0.5 - 1.90 10 100 12 S 42 10 0.26 - 1.46 7 70 13 S 43 11 0.2 - 1.1 8 72.7 14 S 45 4 0.40 -0.96 4 100 15 S 47 9 0.21 - 0.84 5 55.5 16 S 48 9 0.22 - 1.0 1 11.1 17 S 51 6 0.28 - 0.80 2 33.3 18 S 60 6 0.16 - 1.30 6 100 WNRF Technical Bulletin : 12 39 WNRF Technical Bulletin : 12 40 WNRF Technical Bulletin : 12 41

Dendrogram based on Nei’s (1972) Genetic distance : method = UPGMA – modified from Neighbour procedure of PHYLIP version 3.5 (1000 replicate boot strapping)

The amplification profiles of 15 Morinda citrifolia (noni) varieties were noticed with 18 primers out of the 60 primers used. Polymorphisms were observed with fifteen out of these eighteen primers. The banding patterns produced with one primer were not polymorphic for any of the 15 varieties. The eighteen primers studied amplified a total of 102 DNA fragments. Out of 108 bands generated, 80 were polymorphic (74.07 % polymorphism). On an average, the primers generated 5.7 products and 4.4 polymorphisms per primer. The highest number of amplified fragments was 11 by primer S 43 out of which 8 bands were polymorphic (72.7%). The least number of amplified fragments were noticed by S 5 and S 6(one band) showing no polymorphism between any species. The number and size of amplified fragments also varied with different primers. A maximum of 10 fragments were amplified with primer S 41 and all were found to be polymorphic. The size of amplified fragments also varied with different primers. This might be indicative of a narrow genetic base for some of the Morinda varieties studied. The levels of polymorphism were different with different primers among different species. The size of the fragments varied between 0.2 Kb – 1.3 Kb.

In the dendrogram, RHT56006 (RHT garden) sample does not cluster with any other species tested and is easily distinguishable but very closely related to RHT56039, RHT56047, RHT56048 and RHT 56049 (group III). RHT56039 (collected from Mala, in Kerala) is also quite distinct from rest of the species. WNRF Technical Bulletin : 12 42

The intra special polymorphisms detected by RAPD analysis suggest that the species is not homogeneous and segregation might still be occurring in the population. But the similarity matrix shows that there is no much polymorphism between the various accessions of Morinda taken for analysis. The maximum similarity is seen between many species like RHT 56044, RHT 56045 & RHT56046 (0.98) and minimum similarity values (0.82) between RHT 50006 & RHT 56056 which indicates low genetic polymorphisms between the species tested. This may be due to the propagation modules of the plant where propagation may be mostly within a closed area. Most of the seeds are not viable. Some propagate through seed dispersal by birds (Personal communication). The Group I and Group II are closely related probably due to ecological adaptations as they are found closer to the sea shore. Many variations are encountered in these species when compared to the riverine varieties. These varieties might have developed salt tolerance. (Personal communication) These results are a good indication of the reliability of the RAPD technique for the evaluation of genetic similarities. Similarity matrix data also revealed few species RHT 56043 RHT 56044 and RHT560045 showing very few polymorphisms and are closely identical. Interestingly, the first two varieties are in a private land grown under protected environment and the latter is directly exposed to sea winds. On the basis of the RAPD data their genetic base looks very narrow. Intra species studies describe the usefulness of the technique for the evaluation of seed purity and of the genetic similarity of the Morinda varieties. Moreover, the genetic similarities obtained from the analysis can be used for the selection of parents to generate a mapping population and will also help in the selection of parents for breeding purposes. cpDNA Regions Analyzed

The trnL (UAA) intron and the intergenic spacer between the trnL (UAA) 3’ exon and the trnF (GAA) gene was amplified and sequenced with primers c and d and e and f, respectively (Taberlet et al. 1991). The rbcL gene was amplified and sequenced with primers rbcL F and rbcL R. WNRF Technical Bulletin : 12 43

Table-66: Sequencing primers for the non-coding regions located between trnT and trnL gene in plants

S. No cpDNA Regions Primers Expected Size Analyzed of the amplicon (bp)

1. trnL (UAA) intron Tab c and d Sequencing inadequate

2. rbcL rbcL F and rbcL R Sequencing inadequate

3. Intergenic spacer Tab a and b 761bp between the trnT- trnL

Table-67: Sequence of primers used.

Primers Sequence Tab c 5'- CAT TAC AAA TGC GAT GCT CT-3 Tab d 5'- TCT ACC GAT TTC GCC ATA TC-3' rbcL F 5’- ATG TCA CCA CAA ACA GAA AC- 3’ rbcL R 5’- TCG CAT GTA CCT GCA GTA GC - 3’ Tab a 5’- CAT TAC AAA TGC GAT GCT CT – 3’ Tab b 5’- TCT ACC GAT TTC GCC ATA TC -3’

BARCODING Morinda citrifolia var. bracteata voucher RHT56006 tRNA-Thr (trnT) gene, partial sequence; trnT-trnL intergenic spacer, complete sequence; and tRNA-Leu (trnL) gene, partial sequence; chloroplast

GenBank: HM246519.1 LOCUS HM246519 761 bp DNA linear PLN 04-JUL-2010 DEFINITION Morinda citrifolia voucher RHT56006 tRNA-Thr (trnT) gene, artial sequence; trnT-trnL intergenic spacer, complete sequence; andtRNA- Leu (trnL) gene, partial sequence; chloroplast. WNRF Technical Bulletin : 12 44

ACCESSION HM246519 VERSION HM246519.1 GI:299800706

KEYWORDS . SOURCE chloroplast Morinda citrifolia (Indian mulberry) ORGANISM Morinda citrifolia

Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core eudicotyledons; asterids; lamiids; Gentianales; Rubiaceae; Rubioideae; Morindeae; Morinda. REFERENCE 1 (bases 1 to 761) AUTHORS John Britto,S., Manimala,N., Joelri Michael Raj,L. and Senthilkumar,S.R. TITLE Molecular characterization of Morinda citrifolia from different ecological habitats of South India JOURNAL Unpublished REFERENCE 2 (bases 1 to 761) AUTHORS John Britto,S., Manimala,N., Joelri Michael Raj,L. and Senthilkumar,S.R. TITLE Direct Submission JOURNAL Submitted (14-MAY-2010) The Rapinat Herbarium and Centre for Molecular Systematics, St. Joseph’s College, Teppakulam, Tiruchirappalli, Tamil Nadu - 620002, India FEATURES Location/Qualifiers source 1..761 /organism=”Morinda citrifolia” /organelle=”plastid:chloroplast” /mol_type=”genomic DNA” WNRF Technical Bulletin : 12 45

/specimen_voucher=”RHT56006" /db_xref=”taxon:43522" /country=”India: RHT garden, Tiruchirappalli” /lat_lon=”10.8028 N 78.6986 E” /identified_by=”S. John Britto” /note=”PCR_primers=fwd_name: tab a, rev_name: tab b” ORIGIN 1 ATCCATTTTA TATCCAGAGG AATTCAATAA ACTATTAGCA TCTTAGTTAT TAACTAACTA 61 TTTCTTATCT AGTCAAATAG TCAAAGTCGA TATGAATTGA ATATAGAAAA TAATAGAATT 121 TCAAATCAAT ATTGAATATT ATAGAACACA ACGATTAATA GAACGATATA GAATTTCGAT 181 TTATTTATCA CTAATACAAT TCGAATATAA TATTATTAAA TTTGATATAT CTTTTATTAT 241 TTTATTTTTG AGTTTTAGAT AATTAAAGTT TTCCTTTTTG AATTCAAATG ACTTTTGAAC 301 CTCTTTTTAT TGTATTATTT TATTTGATTC TATATCATCG ATTTTATTTC GAATTACGAA 361 TGGTTAGTTA TAACTAATGA GACATTATCC ATTTTCAGTC ATAAAGAGGT AAGTCGGAAA 421 GGCGGAAATT AAGACGACAG AAAGAGATTC GACCCTTCAA GTATTCAAAA TTACATCGAA 481 AATTTGACAG TTAGATCGAT ATACCTAGGA TGATATCTAT CCATCTATAT TGAATTGCGG 541 ATACAGAAAT GGTACAATCA TAATCGATTG GACCAAATCC AAATAGGGTA GGGTTCTCCT 601 ATAGAAAATA GGTAAAAAAT AGGTAATAAG AAGGTAGGTA AGAGATAAAA GAAAACACTT 661 TTTCGAGATA GGAATCCGGT ATTTAATGAA TTCAACGATT CCAGTATAAA TGAAAGAACA 721 AAAGAAACGA CATCACAATG AAAATGAAAT TCGAATCTCA A // Identification of Morinda citrifolia using NCBI Blast 6. Summary and conclusion Intensive field trips and collections of M.citrifolia and its allies chiefly from two Southern states (TamilNadu and Kerala) has revealed several interesting features. Natural populations of M. citrifolia show varied levels of eco types / morphotypes. Depending on the location of the population the habit of M. citrifolia ranges from a foliose shrub to a medium sized tree. The latter forms are mostly seen in sheltered fields and private campuses. They thrive well in coconut plantations sometimes becoming a revenue yielding inter crop. WNRF Technical Bulletin : 12 46

In the interior regions of Kerala the populations is dwindling because human interference. Along the coast and river beds there are luxuriant populations. Whereever the coast is very near to the villages there is total depletion of Noni population. Thiruvananthapuram district is a notable example of this phenomenon. The populations of Noni in Kasaragod district deserve a special mention. They have almost colonized the entire coastal strip almost wiping out other plant formations. The union territory of Mahe has elements of exotic origin perhaps owing to its colonial past. An interesting solitary tree in the park is a witness to the above. Similarly a solitary tree adjacent to the small town of Vadasary Konam has established itself as a naturalized tree though it has been planted from Malaysia. The fruits of this tree likewise the leaves are exceptionally longer and larger. During this project which commenced in May 2009 till 2012 January 60 field trips have been undertaken with a collection of 1101 voucher specimens. Following standard taxonomic procedures they have been processed, mounted, accessioned and identified. One set of specimens is with WNRF, Chennai. The other is with the parent institution namely the Rapinat Herbarium, St. Joseph’s College (Autonomous), Tiruchirappalli. Another notable feature is the field label pasted in the mounting sheet providing location, district, state and the lat. long. Brief notes on the ecology and the population are special levels of information on the population represented by the voucher specimen. In the Final and completion report all the data pertaining to the taxonomic work has been amply provided. To add completion to the project aspect such as molecular systematics, phytochemical analysis, anti-inflametry activity antioxident analysis etc. also have been completed. Besides these several unique photographs of the natural populations and their pheynotypes have found a place in the completion report. The result of this project is from a reputed internationally recognized herbarium and the results of the project will prove a valuable source material to other research endevours financially supported by WNRF. This project has revealed more facets on peninsular Noni populations. WNRF Technical Bulletin : 12 47

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