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Documentation of Orchid Resources of Kiphire, Tuensang and Zunheboto Districts of Nagaland and Micropropagation of Two Threatened Species

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Documentation of Orchid Resources of Kiphire, Tuensang and Zunheboto Districts of Nagaland and Micropropagation of Two Threatened Species Documentation of Orchid Resources of Kiphire, Tuensang and Zunheboto Districts of Nagaland and Micropropagation of Two Threatened Species Submitted By Mr. Hutoka Y. Jakha THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT OF THE DEGREE OF DOCTOR OF PHILOSOPHY IN BOTANY DEPARTMENT OF BOTANY NAGALAND UNIVERSITY, LUMAMI 798 627 NAGALAND, INDIA 2018 1 Contents Chapters Pages Contents 2 Declaration 3 Acknowledgement 4 List of Tables 5-6 List of Figures 7-8 List of Colour Plates 9-11 Ph. D. Course Work Mark Sheet and Certificate 12 Chapter 1: Introduction 13-45 Chapter 2: Documentation 46-283 Introduction 46-47 Materials and Methods 47-59 Taxonomic Treatment 59-268 Results 269-276 Discussion 276-282 Summary and Conclusion 283 Chapter 3: Micropropagation 284-343 Introduction 284-285 Materials and Methods 285-289 Results 289-329 Discussion 330-343 Summary and Conclusion 343 Chapter 4: Summary 344-348 References 349-375 List of Publications 376 Paper Presented in Seminars/Conferences 377 2 3 4 List of Tables Table No. Table Legend Page No. 2.1 G.P.S points of the areas and localities of the three districts 57-58 visited during field survey 2.2 List of orchid species documented from the three districts 270-274 viz., Kiphire, Tuensang and Zunheboto 2.3 Orchid genera represented by number of species and its 274-276 habitat 3.1 Effect of green pod age on in vitro germination of 290 Paphiopedilum insigne. 3.2 Effect of green pod age on in vitro germination of 290 Paphiopedilum villosum var. boxallii. 3.3 Effects of nutrient media on non-symbiotic seed germination 296 of P. insigne 3.4 Effects of different nutrient media on non-symbiotic seed 297 germination of P. villosum var. boxallii. 3.5 Effect of different organic carbon sources on in vitro culture 298 of immature embryos of P. insigne. 3.6 Effect of different organic carbon sources on in vitro embryo 299 culture of P. villosum var. boxallii. 3.7 Effect of coconut water (CW) on non-symbiotic seed 300 germination of P. insigne. 3.8 Effect of CW on non-symbiotic seed germination of P. 300 villosum var. boxallii. 3.9 Effects of activated charcoal (AC) on non-symbiotic seed 301 germination of P. insigne. 3.10 Effects of activated charcoal (AC) on non-symbiotic seed 301 germination of P. villosum var. boxallii. 3.11 Effect of different levels of PGRs on non symbiotic seed 304 germination of P. insigne. 3.12 : Effect of quality and quantity of PGRs on non symbiotic 305 seed germination of P. villosum var. boxallii. 3.13 Effect of PGRs on morphogenetic response of foliar explants 309 (̴ 8 wk old) of P. insigne from in vitro source. 3.14 Effect of PGRs on morphogenetic response of foliar explants 310 (̴ 8 wk old) of P. villosum var boxallii from in vitro source. 3.15 Effect of nutrient media on regeneration of plantlets of 313 Paphiopedilum insigne. 3.16 Effect of different basal media on regeneration of plantlets 313 of Paphiopedilum villosum var boxallii. 3.17 Effect of different strengths of MS medium for PLBs 315 differentiation, plant regeneration and mass multiplication of Paphiopedilum insigne. 3.18 Effect of strengths of MS medium for PLBs differentiation, 315 plant regeneration and mass multiplication of Paphiopedilum villosum var. baoxallii. 3.19 Effect of different organic carbon sources on plant 316 regeneration and mass multiplication of Paphiopedilum 5 insigne. 3.20 Effect of different organic carbon source on plant 317 regeneration and mass multiplication of Paphiopedilum villosum var. boxallii. 3.21 Effect of different PGRs on plant regeneration and mass 319-320 multiplication of Paphiopedilum insigne. 3.22 Effect of different PGRs on plant regeneration and mass 321-322 multiplication of Paphiopedilum villosum var boxallii. 3.23 Percentage survival of in vitro grown plantlets of 328 Paphiopedilum insigne and Pahiopedilum villosum var boxallii in different potting mix 6 List of Figures Figure Figure Legend Page No. 1.1 Paphiopedilum villosum var. boxallii Wild Flowering and 41 close up: a. Dorsal sepal; b. Petals; c. Lip; d. Synsepal; e. Ovary with pedicel and bract; f. Column; g. Seed pod. 1.2 Paphiopedilum insigne Plants and Close up flower: a. Dorsal 42 sepal; b. Petals; c. Lip; d. Synsepal; e. Ovary with pedicel and bract; f. Column; g. Seed pod. 1.3 Forest Cover Map of Nagaland (Source: Department of 45 Forest, Ecology, Environment and Wildlife; Nagaland). 2.1 Map of Study area (Not to scale). 49 2.2 Forest Cover Map of Kiphire District, Nagaland (Source: - 50 Department of Forest, Ecology, Environment and Wildlife, Nagaland). 2.3 Forest Cover Map of Tuensang District, Nagaland (Source:- 51 Department of Forest, Ecology, Environment and Wildlife, Nagaland). 2.4 Forest Cover Map of Zunheboto District, Nagaland (Source:- 52 Department of Forest, Ecology, Environment and Wildlife, Nagaland). 2.5 Excerpt from field studies ; a. Logging at Helipong range 281 forest, Tuensang district ; b. Collection of orchids from a logged tree; c. At base camp Wangsoi village; d. Field guide helping in collection of wild Paphiopedilum villosum var. boxallii - Indo-Myanmar border Tuensang; e. Returning from field work, Tsutoho village, Zunheboto; f. Field collection for later sorting; g. Indo-Myanmar Border stone bordering DAN ITC, Tuensang district; h. Indo-Myanmar boundary stone atop Saramati range, Kiphire district. 2.6 Panoromic view of Jhumming cultivation; a. Fresh Slash and 282 burn (Jhum) for cultivation at Noklak area, Tuensang; b. Large forest areas converted for Jhum cultivation, Longmatra, Kiphire district; c. Cultivation after Jhumming, V.K area, Zunheboto. 3.1 Green pod morphology at various developmental age of 293 P.aphiopedilum insigne. a. 180 DAP (Days after pollination) (Immature green pods); b. 210 DAP (Immature green pods); c. 240 DAP (Immature green pods); d. 270 DAP(Fully mature pods) and e. 300 DAP (Burst out seed pods with microscopic seeds). 3.2 Different developmental ages and morphology of green pods 294 of Paphiopedilum villosum var. boxallii. a. 210 DAP (Immature green pods); b. 240 DAP (Immature green pods); c. 270 DAP (Mature seed pods); d. 360 DAP (Burst out seed pods with microscopic seeds). 3.3 Orchid seeds at various stages of culture. a-c: P. insigne and 295 7 d-f: P. villosum var. boxallii. a. Stained seeds before inoculation; b. Stained viable seeds showing embryo and seed coat; c. Nodular swelling of embryos from 210 DAP pods; d. Stained seeds before inoculation; e. Stained viable seeds showing embryo and seed coat; f. Nodular swelling of embryos from 240 DAP. 3.4a Effect of light on in vitro culture of immature embryo of P. 302 insigne. 3.4b Effect of light on in vitro culture of immature embryo of P. 302 villosum var. boxallii. 3.5 a. PLBs formation Paphiopedilum insigne; b. PLBs 307 formation Paphiopedilum villosum var. boxallii ; c. Micro plants with leaf initials of P. insigne and d. Micro plants with leaf initials of P. villosum var. boxallii. 3.6 Different stages of in vitro morphogenetic response from 308 foliar explants a. Shoot buds developed from foliar explants of P. insigne b. Multiple shoot growth P. insigne c. Shoot buds developed from foliar explants of P. villosum var. boxallii, d. Multiple shoots developed from foliar explants of P. villosum var. boxallii. 3.7 Plantlets a. P. insigne with root and shoot but no multiple 311 shoot buds b. P. villosum var. boxallii with root and shoot but no multiple shoot buds c. P. insigne abnormal root growth with multiple shoots. 3.8 Plant regeneration and culture proliferation. a and b. P. 323 insigne; c and d. P. villosum var. boxallii. 3.9 Different stages plant regeneration of P. insigne. a. Advance 324 stage PLBs/shoot buds on regeneration medium, b. Regenerated plantlets, c. Well rooted plantlet ready for hardening. 3.10 Different stages plant regeneration of P. villosum var 326 boxallii a. Advance stage PLBs/shoot buds on regeneration medium, b. Regenerated plantlets, c. Fully formed plantlet ready for hardening. 3.11 Different types of hardening of regenerates of P. insigne and 327 P. villosum var. boxallii. a. Coconut husk (P. insigne); b, c. Brick pieces, charcoal chunks, coconut coir (at 1:1:1 ratio) (P. insigne and P. villosum var. boxallii); d. Sand + Decaying organic matter (1:1) (P. villosum var. boxallii). e, f. Wooden pieces (P. insigne and P. villosum var. boxallii); g. Sand + Decaying organic matter + brick pieces + Charcoal + Dried cow dung (1:1:1:1) (P. insigne and P. villosum var. boxallii); h. Moss(P. villosum var. boxallii). 3.12 Hardened regenerates estalished in potting mix ready for 329 field transfer. Plants in the Nursery a. P. insigne, b. P. villosum var. boxallii 8 Colour Plates Plate No. Plate Legend Page No. 1 a) Acampe praemorsa (Roxb.) Blatt. & Mc Cann; b) Acampe 239 rigida (Buch.-Ham. ex Sm.) P.F.Hunt; c) Acampe ochracea (Lindl.) Hochr.; d) Acanthephippium striatum Lindl.; e) Aerides multiflora Roxb.; f) Aerides odorata Lour. 2 a) Agrostophyllum. callosum Rchb.f. b) Anthogonium gracile 240 Lindl.; b) Acampe rigida (Buch.-Ham. ex Sm.); c) Arachnis labrosa (Lindl. ex Paxt) Rchb.f.; d) Arachnis labrosa var zhaoi.; e) Arachnis senapatiana (Phukan & A.A. Mao) Kocyan & Schuit.; f) Arundina graminifolia (D. Don) Hochr. 3 a) Bulbophyllum affine Lindl.; b) Bulbophyllum candidum 241 Hook.f.; c) Bulbophyllum caryanum (Hook.) Spreng; d) Bulbophyllum cariniflorum Rchb. f.; e) Bulbophyllum crassipes Hook. f.; f) Bulbophyllum cylindraceum Lindl. 4 a) Bulbophyllum elatum (Hook.f.) J.J.Sm.; b) Bulbophyllum. 242 helenae ( Kuntze.) J. J. Sm.; c) Bulbophyllum leopardinum (Wall.) Lindl. ;d) Bulbophyllum odorotissimum (J.E. Smith) Lindl.; e) Bulbophyllum pteroglossum Schltr.; f) Bulbophyllum reptans Lindl.
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