DOCSLIB.ORG

[Paphiopedilum Niveum (Rchb.F.) Stein] Using V Cryo-Plate Method

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
[Paphiopedilum Niveum (Rchb.F.) Stein] Using V Cryo-Plate Method วารสารพืชศาสตร์สงขลานครินทร์ ปีที่ 6 ฉบับที่ 4 (ตุลาคม-ธันวาคม): 10-18, 2562 Songklanakarin Journal of Plant Science, Vol. 6, No. 4 (October-December): 10-18, 2019 Research article The Investigation of Condition for Cryopreservation of Snow-White Venus’s Slipper Orchid Protocorm [Paphiopedilum niveum (Rchb.f.) Stein] using V cryo-plate Method Soonthornkalump, S.1* Yamamoto, S.2 Nakkanong, K.3 and Meesawat, U.1 1 Department of Biology, Faculty of Science, Prince of Songkla University, Kho Hong, Hat Yai, Songkhla, Thailand, 90110 2 Genetic Resources Center, National Agriculture and Food Research Organization (NARO), 2-1-2 Kanondai, Tsukuba, Ibaraki, Japan, 305-8518 3 Department of Plant Science, Faculty of Natural Resources, Prince of Songkla University, Kho Hong, Hat Yai, Songkhla, Thailand, 90110 * Corresponding author: [email protected] Received 26 April 2019; Revised 24 May 2019; Accepted 11 June 2019 Abstract Paphiopedilum niveum (Rchb.f.) Stein is native orchid to Southern Thailand which has been considered as an endangered species. The over collection decreased its wild population, so conservation of its genetic material is needed. The cryopreservation is efficient long term storage method. The objective of this study focused on the investigation of sucrose concentration at the preculture step and V cryo-plate protocol development to cryopreserved protocorm of P. niveum. In order to investigate the optimal sucrose concentration in the preculture medium , two-month-old protocorms were precultured in modified Vacin and Went medium (MVW) containing 0.058, 0.2, 0.4, 0.6 and 0.8 M sucrose for 24 h. The viability determination was tested by TTC assay and anatomical observation. The result presented that precultured protocorms in 0.2 M sucrose provided the highest survival at 96% without anatomical damage which was placed as the 1st preculture step. The highest survival percentage (60%) of non-cryopreserved protocorm was obtained from procedure followed by the 1st preculture in MVW containing 0.2 M sucrose (1 day) and 0.6 M sucrose (1 day), followed by osmoprotection using loading solution (LS) containing 1.2 M sucrose (30 min). Protocorms were dehydrated by PVS2 incubation for 60 min. However, there was no survival of cryopreserved protocorm after preserved in liquid nitrogen. Keywords: Anatomical observation, endangered species, genetic resources conservation, orchid Introduction Paphiopedilum niveum (Rchb.f.) Stein is the Although wild populations have been conserving in terrestrial orchid which distributed on the shading area the national park, the illegal poaching is still threating. of limestone of the Northern Peninsular Malaysia and Recently, the ex situ conservation could be conserved Andaman archipelago (Pedersen et al., 2011). The a number of P. niveum in botanic garden as living conservation status of P. niveum was evaluated to specimens. However, the conservation using endangered (EN) by the International Union for cryopreservation may useful in long term conservation Conservation of Nature (IUCN) because the continuous program. Cryopreservation is the technique for long- decrease of wild population from human intrusions term storage under extremely low temperature. and disturbance (Pedersen et al., 2011; Rankou, 2015). Generally, the cryopreservation method usually Online open access e-journal : www. natres.psu.ac.th/department/plantScience/sjps/default.htm Published by Department of Plant Science, Faculty of Natural Resources, Prince of Songkla University. All rights reserved. For Permissions, please e-mail: [email protected]. Soonthornkalump et al. (2019) consisted of five major steps which are preculture, of P. niveum protocorm is still under development. osmoprotection, dehydration, storage in liquid This study was carried out to develop the V cryo-plate nitrogen (LN), and regrowth. In cryopreservation, the protocol for cryopreserved P. niveum protocorm. actively meristematic tissues or cells are required such as, shoot tips and somatic embryo (Sakai and Materials and methods Engelmann, 2007). Samples are generally excised from Plant material preparation mother plant into the small size which allowed the Five-month-old P. niveum capsule was good penetration and absorption of cryoprotectant surface sterilized by dip into 70% ethanol then during the cryopreservation (Niino et al., 2017). flame for a few second to diminish hairs. Flamed Preculture step induces the endogenous capsule was longitudinal excised and seeds were cryoprotectant accumulation (Kaczmarczyk et al., transferred to sterile distilled water for 2 weeks. 2012). Loading treatment containing glycerol and Then, seeds were transferred to modified liquid sucrose increase the cellular osmolality which induces Vacin and Went medium (MVW) (Vacin and Went, tolerance to freezing dehydration (Reed, 2008). Plant 1949) containing full strength VW macronutrient and vitrification solution 2 (PVS2) is a high concentrated half strength MS (Murashige and Skoog, 1962) vitrification solution which is used in dehydration micronutrient supplemented with 5 mg/L chitosan, before rapid immersion in LN (Sakai and Engelmann, 2 g/L peptone and 2% (w/v) sucrose. The pH of 2007). medium is adjusted to 5.3 with 1 N NaOH. Medium Preculture is the first important step that was sterilized with autoclave at 121 °C for 20 min. needs to determine in cryopreservation protocol. In Seeds were cultured under dark condition on 120 addition, a physiological response during preculture rpm orbital agitator at 25±2 °C. Two-month-old step can be occurred. For instance, the increasing of protocorms (0.5-1 mm diameter) with shoot pole abscisic acid (ABA) trigger the proline accumulation in were used as plant materials in the determination plant cells which enhance the desiccation tolerance of appropriated condition of the 1st preculture and (Suzuki et al., 2006). Sucrose in preculture medium V cryo-plate protocol for cryopreservation act as osmolyte which enhances frost hardiness during investigation. cold acclimatization by increase cell membrane integrity (Pinker et al., 2009a). Moreover, the absorbed 1. Influence of the sucrose concentration in sucrose could be converted to the storage form of preculture: viability and histological observation carbohydrate which important in recovery growth Two-month-old protocorms were precultured (Pinker et al., 2009b). Thus, the optimization of in 50 ml liquid MVW medium supplemented with sucrose concentration is essential to determine the various sucrose concentrations (0.058 (control), 0.2, suitable preculture condition in this study. 0.4, 0.6 and 0.8 M) for 24 h under light condition at V cryo-plate method which is developed 25±2 °C on 100 rpm agitator. After incubation, from encapsulation vitrification and droplet vitrification viability of precultured protocorm was determined is the efficient cryopreservation method (Yamamoto et using triphenyltetrazolium chloride (TTC test) which al., 2011). This method can reduce the loss of plant was slightly modified from Verleysen et al. (2004). materials during the processing and is easy to carry on TTC solution containing 0.6% TTC with 0.05% Tween (Sekizawa et al., 2011). V cryo-plate was applied to 85 in a 0.05 M Na2HPO4/KH2PO4 buffer and the pH conserve many plant species such as Clinopodium was adjusted to pH 7.4. Four protocorms were odorum (Griseb.) Harley (Engelmann-Sylvestre and placed into a centrifuge tube containing 1.5 ml of Engelmann, 2015), Dianthus caryophyllus L. (Sekizawa TTC solution and incubated overnight at room et al., 2011) and Morus spp. (Yamamoto et al., 2011). temperature. The viable protocorms showing red However, an optimized protocol for cryopreservation stained color could be visualized under microscopic 11 ว. พืชศาสตร์สงขลานครินทร์ 6 (4): 10-18 Songklanakarin J. Pl. Sci., 6 (4): 10-18 Soonthornkalump et al. (2019) observation. Three replicates, each with ten then dropped for gel hardening (15 min). After that, protocorms, were used in each treatment. Samples the cryo-plate with embedded protocorms were of precultured protocorms were fixed with FAA II transferred to osmoprotection step by immersed (formaldehyde: glacial acetic acid: 70% ethanol; into loading solution (LS) containing 2 M glycerol 5:5:90 v/v/v) for 48 h. Fixed protocorms were rinsed with sucrose (0.4, 0.8 and 1.2 M) for 30 min. These and stored with 70% ethanol. Fixed protocorms were protocorms were dehydrated in PVS2 containing dehydrated using tertiary-butyl-alcohol (TBA) series at 30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% 70%, 85%, 95% and 100%, 2 h for each (w/v) DMSO and 0.4 M sucrose (Sakai and concentration. After that, samples were immersed in Engelmann, 2007) at different exposure times (for the mixture of paraffin oil : paraffin wax, 1:1 (v/v) 30, 45 and 60 min). The cryo-plate was put into followed by soaked in pure paraffin wax (Histoplast) cryotube and then plunged in LN at least for 1 h. for infiltration (2 h for each step at 56 °C in the hot air oven) (Ruzin, 1999). A piece of sample was Rewarming and survival determination embedded in paraffin wax. Embedded specimens Cryopreserved protocorms were rewarmed were cut into 6 µm thickness with a rotary in 1 M sucrose solution at room temperature for 15 microtome (AO, 820 SPENCER). The section was min (Sekizawa et al., 2011). The post-rewarming affixed to glass slide and stained with Delafield’s protocorms were cultured in iron-free solid MVW hematoxylin and safranin staining to observe the medium containing 0.1 mg/L 1-Naphthaleneacetic histological alteration (Johansen, 1940). Periodic acid (NAA), 0.2% (w/v) polyvinylpyrrolidone (PVP-40) acid–Schiff (PAS) reaction was employed to examine and 0.2 % (w/v) activated charcoal (AC) under the the storage carbohydrate (Ruzin, 1999). The darkness at 25±2 °C for 7 days and then transferred preculture medium containing optimal sucrose to light condition. After 1 month of culture, survival concentration that provided the high survival percentage was determined using visual percentage with the less histological and observation. The viable lutescent and non-viable histochemical changes would be used in the further browning protocorms were determined as survival experiment.
Load more

Recommended publications
  • The Orchid Society of Karnataka (TOSKAR) Newsletter – June 2016 1
    The Orchid Society of Karnataka (TOSKAR) Newsletter – June 2016 1 The Orchid Society of Karnataka (TOSKAR) Newsletter – June 2016 2 The Orchid Society of Karnataka (TOSKAR) Newsletter – June 2016 3 NAGESHWAR’S JOURNEY FROM ONION TO ORCHIDS Dr N. Shakuntala Manay Here is Nagesh’s story, the first recipient of TOSKAR Rolling Shield for the Best Orchid. His interest in growing plants started as a child of eight when he would pick up sprouting onions from Mom’s kitchen onion and plant them in the yard and watched them grow into green leeks. This got him into the hobby to grow vegetables. By this time he was 14. Later he turned to growing foliage plants like succulents, Anthuriums and Cacti. Thus he dared to enter into annual shows at Lalbagh and won many prizes. In “small homes garden” categories he won eight awards from Urban Art Commission such as “Best Maintained Building & Garden” “Pride of Bangalore” “Role of Honour” etc. Ex- commissioners of Bangalore City Corporation Late N. Laxman Rao and Late Mr. Parthsarathy would visit his house as Judges. He received these prestigious prizes amidst distinguished guests and dignitaries at Rajbhavan. Trophies gathered so fast that there was no place for them at home. Twenty years ago he got one orchid from Indo American Nursery. Thus he began collecting orchids from Kerala, North East India and Western Ghats. Now on his terrace of 800 sq ft he has 1500 orchids! Among these Dracula Orchid (Monkey face) which grows in cloud mountains of Mexico, Central America and Colombia is one of his special collections, and more than 15 varieties of Carnivorous Plants and many Tillandsias also add to his collection.
    [Show full text]
  • Index Sorted by Title
    Index sorted by Title Volume Issue Year Article Title Author Key Words 31 5 1967 12th Western Orchid Congress Jefferies, George Western Orchid Congress 31 5 1967 12th Western Orchid Congress — Photo Flashes Philpott, R. G. Western Orchid Congress 12th World Orchid Conference ... March 1987, 51 4 1987 Eilau, William World Orchid Conference, Tokyo Tokyo, Japan 13th World Orchid Conference, Auckland, New World Orchid Conference, New 54 2 1990 Eilau, William Zealand Zealand 14th World Orchid Conference, Glascow, 57 3 1993 Hetherington, Ernest World Orchid Conference, scotland Scotland, April 26-May 1, 1993, The 1992 Volume of the Orchid Digest is Dedicated 56 1 1992 in Memoriam to D. George Morel (1926-1973), Hetherington, Ernest history, George Morel The 58 4 1994 1994 Orchid Digest Research Grant Digest Staff 1994 orchid, research, grant 1995 Orchid Digest Dec Dedicated to Herb 59 1 1995 Digest Staff Dedication, Herb Hager Hager 72 2 2008 19th World Orchid Conference Hersch, Helen world orchid conference, 19th 2018 Paphiopedilum Guild and the Second 2018, paphiopedilum guild, second 82 2 2018 International World Slipper Orchid Conference Sorokowsky, David international world slipper orchid, Hilo, Hawaii conference 80 3 2016 22nd World Orchid Conference Pridgeon, Alec 22nd World Orchid Conference 84 4 2020 A Checklist of Phramipedium Species Cervera, Frank checklist, phragmipedium 84 3 2020 A New Color Forma for Vanda curvifolia Koopowitz, Harold vanda, curvifolia, new color form A New Species of Lepanthes (Orchidaceae: Larson, Bruno, Portilla, Jose, Medina 85 2 2021 new species, Lepanthes, Ecuador Pleurothallidinae) from South East Ecuador Hugo A New Species of Pleurothallopsis new species, pleurothallopsis, 82 1 2018 (Epidendreae, Epidendroideae, Orchidaceae): Matthews, Luke M.
    [Show full text]
  • CITES Orchids Appendix I Checklist
    CITES Appendix I Orchid Checklist For the genera: Paphiopedilum and Phragmipedium And the species: Aerangis ellisii, Cattleya jongheana, Cattleya lobata, Dendrobium cruentum, Mexipedium xerophyticum, Peristeria elata and Renanthera imschootiana CITES Appendix I Orchid Checklist For the genera: Paphiopedilum and Phragmipedium And the species: Aerangis ellisii, Cattleya jongheana, Cattleya lobata, Dendrobium cruentum, Mexipedium xerophyticum, Peristeria elata and Renanthera imschootiana Second version Published July 2019 First version published December 2018 Compiled by: Rafa¨elGovaerts1, Aude Caromel2, Sonia Dhanda1, Frances Davis2, Alyson Pavitt2, Pablo Sinovas2 & Valentina Vaglica1 Assisted by a selected panel of orchid experts 1 Royal Botanic Gardens, Kew 2 United Nations Environment World Conservation Monitoring Centre (UNEP-WCMC) Produced with the financial support of the CITES Secretariat and the European Commission Citation: Govaerts R., Caromel A., Dhanda S., Davis F., Pavitt A., Sinovas P., & Vaglica V. 2019. CITES Appendix I Orchid Checklist: Second Version. Royal Botanic Gardens, Kew, Surrey, and UNEP-WCMC, Cambridge. The geographical designations employed in this book do not imply the expression of any opinion whatsoever on the part of UN Environment, the CITES Secretariat, the European Commission, contributory organisations or editors, concerning the legal status of any country, territory or area, or concerning the delimitation of its frontiers or boundaries. Acknowledgements The compilers wish to thank colleagues at the Royal Botanic Gardens, Kew (RBG Kew) and United Nations Environment World Conservation Monitoring Centre (UNEP-WCMC). We appreciate the assistance of Heather Lindon and Dr. Helen Hartley for their work on the International Plants Names Index (IPNI), the backbone of the World Checklist of Selected Plant Families. We appreciate the guidance and advice of nomenclature specialist H.
    [Show full text]
  • (Paphiopedilum Niveum (Rchb.F.) Stein) Sutthinut Soonthornk
    In Vitro Propagation and Cryopreservation of Lady’s Slipper Orchid (Paphiopedilum niveum (Rchb.f.) Stein) Sutthinut Soonthornkalump A Thesis Submitted in Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology Prince of Songkla University 2019 Copyright of Prince of Songkla University i In Vitro Propagation and Cryopreservation of Lady’s Slipper Orchid (Paphiopedilum niveum (Rchb.f.) Stein) Sutthinut Soonthornkalump A Thesis Submitted in Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology Prince of Songkla University 2019 Copyright of Prince of Songkla University ii Thesis Title In Vitro Propagation and Cryopreservation of Lady’s Slipper Orchid (Paphiopedilum niveum (Rchb.f.) Stein) Author Mr. Sutthinut Soonthornkalump Major Program Biology Major Advisor Examining Committee: ……………………………………… ..………………………….Chairperson (Assoc. Prof. Dr. Upatham Meesawat) (Assoc. Prof. Dr. Kanchit Thammasiri) ..…………………………..Committee (Assoc. Prof. Dr. Upatham Meesawat) Co-advisor ……………………………………… ..…………………………..Committee (Asst. Prof. Dr. Korakot Nakkanong) (Prof. Dr. Sompong Te-chato) ……………………………………… ..…………………………..Committee (Dr. Shin-ichi Yamamoto) (Asst. Prof. Dr. Korakot Nakkanong) ..…………………………..Committee (Dr. Shin-ichi Yamamoto) The Graduate School, Prince of Songkla University, has approved this thesis as fulfillment of the requirements for the Doctor of Philosophy Degree in Biology. ………………………………………. (Prof. Dr. Damrongsak Faroongsarng) Dean of Graduate School iii This is to certify that the work here submitted is the results of candidate’s own investigations. Due acknowledgement has been made of any assistance received. ….…………………………..Signature (Assoc. Prof. Dr. Upatham Meesawat) Major Advisor ……………………………...Signature (Mr. Sutthinut Soonthornkalump) Candidate iv I hereby certify that this work has not already been accepted in substance for any degree, and is not being concurrently submitted in candidature for any degree.
    [Show full text]
  • Index Sorted by Title
    Index sorted by Title Volume Issue Year Article Title Author Key Words 31 5 1967 12th Western Orchid Congress Jefferies, George Western Orchid Congress 31 5 1967 12th Western Orchid Congress — Photo Flashes Philpott, R. G. Western Orchid Congress 12th World Orchid Conference ... March 1987, 51 4 1987 Eilau, William World Orchid Conference, Tokyo Tokyo, Japan 13th World Orchid Conference, Auckland, New World Orchid Conference, New 54 2 1990 Eilau, William Zealand Zealand 14th World Orchid Conference, Glascow, 57 3 1993 Hetherington, Ernest World Orchid Conference, scotland Scotland, April 26-May 1, 1993, The 1992 Volume of the Orchid Digest is Dedicated 56 1 1992 in Memoriam to D. George Morel (1926-1973), Hetherington, Ernest history, George Morel The 58 4 1994 1994 Orchid Digest Research Grant Digest Staff 1994 orchid, research, grant 59 1 1995 1995 Orchid Digest Dec Dedicated to Herb Hager Digest Staff Dedication, Herb Hager 72 2 2008 19th World Orchid Conference Hersch, Helen world orchid conference, 19th 2018 Paphiopedilum Guild and the Second 2018, paphiopedilum guild, second 82 2 2018 International World Slipper Orchid Conference Sorokowsky, David international world slipper orchid, Hilo, Hawaii conference 80 3 2016 22nd World Orchid Conference Pridgeon, Alec 22nd World Orchid Conference 84 4 2020 A Checklist of Phramipedium Species Cervera, Frank checklist, phragmipedium 84 3 2020 A New Color Forma for Vanda curvifolia Koopowitz, Harold vanda, curvifolia, new color form A New Species of Pleurothallopsis (Epidendreae, new species, pleurothallopsis, 82 1 2018 Epidendroideae, Orchidaceae): Pleurothallopsis Matthews, Luke M. alphonsiana alphonsiana 82 3 2018 A Visit to Colombian Cattleyas Popper, Helmut H.
    [Show full text]
  • CITES and Slipper Orchids
    CITES and Slipper Orchids An introduction to slipper orchids covered by the Convention on International Trade in Endangered Species Written by H. Noel McGough, David L. Roberts, Chris Brodie and Jenny Kowalczyk Royal Botanic Gardens, Kew United Kingdom The Board of Trustees, Royal Botanic Gardens, Kew 2006 © The Board of Trustees of the Royal Botanic Gardens, Kew 2006 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form, or by any means, electronic, mechanical, photocopying, recording or otherwise, without written permission of the publisher unless in accordance with the provisions of the Copyright Designs and Patents Act 1988. First published in 2006 by Royal Botanic Gardens, Kew Richmond, Surrey, TW9 3AB, UK www.kew.org ISBN 1-84246-128-1 For information or to purchase Kew titles please visit www.kewbooks.com or email [email protected] Cover image: © Royal Botanic Gardens, Kew CONTENTS Introduction ..................................................................................................... i Acknowledgements ........................................................................................ ii How to Use this Presentation Pack ............................................................... iii References and Resources ........................................................................ iv-ix Slide Index ................................................................................................. x-xi Slides and speaker’s notes .......................................................................
    [Show full text]
  • Effects of Natural Additives on Asymbiotic Seed Germination and Growth of Endangered Lady’S Slipper Orchid: Paphiopedilum Callosum Var
    108 109 Effects of Natural Additives on Asymbiotic Seed Germination and Growth of Endangered Lady’s Slipper Orchid: Paphiopedilum callosum var. sublaeve ผลของส�รจ�กธรรมช�ติต่อก�รงอกของเมล็ดและก�รเจริญเติบโตของกล้วยไม้ รองเท้�น�รีม่วงสงขล� (Paphiopedilum callosum var. sublaeve) นรารัตน์ วัฒนาพันธ์1, จรัสศรี นวลศรี2, อุปถัมภ์ มีสวัสดิ์1 Nararatn Wattanapan1, Charassri Nualsri2, Upatham Meesawat1 บทคัดย่อ รองเท้านารีม่วงสงขลา (Paphiopedilum callosum var. sublaeve) เป็นกล้วยไม้รองเท้านารีประจำาถิ่นที่อยู่ทาง ภาคใต้ของไทย ซึ่งเป็นพืชใกล้สูญพันธุ์ จึงถูกจัดให้อยู่ในบัญชีแนบท้ายหมายเลข 1 ตามอนุสัญญาไซเตส การทดลองนี้ศึกษา อิทธิพลของสารจากธรรมชาติ คือ กรดไฮยาลูโรนิค (HA) และไคโตซาน ที่มีต่อการงอกของเมล็ด การเจริญของโพรโทคอร์ม และการเพิ่มจำานวนยอดของรองเท้านารีม่วงสงขลา โดยเพาะเลี้ยงเมล็ดในอาหารเหลวสูตร MVW ที่มี HA ความเข้มข้น 0, 0.01, 0.1 และ 1.0 มก/ลิตร หรือไคโตซานความเข้มข้น 0, 1, 5 และ 10 มก/ลิตร พบว่า เมล็ดเริ่มงอกภายใน 45 วัน มีค่า อัตราการงอกสูงสุด 8.34±1.01%, ค่าดัชนีการงอกสูงสุด 0.16±0.02, และอัตราการเกิดโพรโทคอร์มสูงสุด 4.28±0.96% เมื่อ เพาะเลี้ยงในอาหารเหลวสูตร MVW ที่มี HA ความเข้มข้น 0.1 มก/ลิตร อย่างไรก็ตาม อาหารวุ้นสูตร MVW ที่มีไคโตซาน ความเข้มข้น 1.0 มก/ลิตร เป็นอาหารที่มีความเหมาะสมต่อการเจริญของโพรโทคอร์มมากที่สุด เนื่องจากให้ค่าอัตราการงอกใน ระยะ C สูงสุด 1.93±0.35% และโพรโทคอร์มที่ได้มีสีเขียวและแข็งแรง ส่วนการเพาะเลี้ยงบนอาหารวุ้นสูตร MMS ที่มี HA ความเข้มข้น 1.0 มก/ลิตร พบว่ามีจำานวนการเกิดยอดสูงสุด 3.22±0.36 ยอด/โพรโทคอร์ม และอัตราการเกิดยอดสูงสุด 100% จากนั้นย้ายต้นที่ได้ไปเพาะเลี้ยงในอาหารวุ้นสูตร MMS และย้ายลงปลูกในกระถางที่มีสแฟคนั่มมอสไปอนุบาลในเรือนเพาะชำาเป็น
    [Show full text]
  • Phylogenetics, Genome Size Evolution and Popula- Tion Genetics of Slipper Orchids in the Subfamily Cypri- Pedioideae (Orchidaceae)
    ORBIT-OnlineRepository ofBirkbeckInstitutionalTheses Enabling Open Access to Birkbeck’s Research Degree output Phylogenetics, genome size evolution and popula- tion genetics of slipper orchids in the subfamily cypri- pedioideae (orchidaceae) https://eprints.bbk.ac.uk/id/eprint/40088/ Version: Full Version Citation: Chochai, Araya (2014) Phylogenetics, genome size evolu- tion and population genetics of slipper orchids in the subfamily cypri- pedioideae (orchidaceae). [Thesis] (Unpublished) c 2020 The Author(s) All material available through ORBIT is protected by intellectual property law, including copy- right law. Any use made of the contents should comply with the relevant law. Deposit Guide Contact: email Phylogenetics, genome size evolution and population genetics of slipper orchids in the subfamily Cypripedioideae (Orchidaceae) Thesis submitted by Araya Chochai For the degree of Doctor of Philosophy School of Science Birkbeck, University of London and Genetic Section, Jodrell Laboratory Royal Botanic Gardens, Kew November, 2013 Declaration I hereby confirm that this thesis is my own work and the material from other sources used in this work has been appropriately and fully acknowledged. Araya Chochai London, November 2013 2 Abstract Slipper orchids (subfamily Cypripedioideae) comprise five genera; Paphiopedilum, Cypripedium, Phragmipedium, Selenipedium, and Mexipedium. Phylogenetic relationships of the genus Paphiopedilum, were studied using nuclear ribosomal ITS and plastid sequence data. The results confirm that Paphiopedilum is monophyletic and support the division of the genus into three subgenera Parvisepalum, Brachypetalum and Paphiopedilum. Four sections of subgenus Paphiopedilum (Pardalopetalum, Cochlopetalum, Paphiopedilum and Barbata) are recovered with strong support for monophyly, concurring with a recent infrageneric treatment. Section Coryopedilum is also recovered with low bootstrap but high posterior probability values.
    [Show full text]
  • Paphiopedium Callosum Var. Sublaeve Nararatn Wattanapan a Thesis
    Growth and Genetic Variation of Lady’s Slipper Orchid: Paphiopedium callosum var. sublaeve Nararatn Wattanapan A Thesis Submitted in Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology Prince of Songkla University 2016 Copyright of Prince of Songkla University i Growth and Genetic Variation of Lady’s Slipper Orchid: Paphiopedium callosum var. sublaeve Nararatn Wattanapan A Thesis Submitted in Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology Prince of Songkla University 2016 Copyright of Prince of Songkla University ii Thesis Title Growth and genetic variation of lady’s slipper orchid: Paphiopedilum callosum var. sublaeve Author Mrs. Nararatn Wattanapan Major Program Biology Major Advisor Examining Committee : ............................................................. .......................................... Chairperson (Assoc. Prof. Dr. Upatham Meesawat) (Assoc. Prof. Dr. Kanchit Thammasiri) .............................................Committee Co-advisor (Assoc. Prof. Dr. Upatham Meesawat) ............................................................. .............................................Committee (Assoc. Prof. Dr. Charassri Nualsri) (Assoc. Prof. Dr. Charassri Nualsri) .............................................Committee (Prof. Dr. Sompong Te-chato) ............................................ Committee (Dr. Aslan Hilae) The Graduate School, Prince of Songkla University, has approved this thesis as fulfillment of the requirements for the Doctor of Philosophy
    [Show full text]
  • DNA Barcoding of Endangered Paphiopedilum Species (Orchidaceae) of Peninsular Malaysia
    Phytotaxa 387 (2): 094–104 ISSN 1179-3155 (print edition) https://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2019 Magnolia Press Article ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.387.2.2 DNA Barcoding of Endangered Paphiopedilum species (Orchidaceae) of Peninsular Malaysia MICHEAL C. RAJARAM1, CHRISTINA S.Y. YONG1, JUALANG A. GANSAU2 & RUSEA GO1* 1Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor Darul Ehsan, Malaysia. 2Faculty of Science and Technology, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia. *Author for correspondence: [email protected] Abstract In this study, the efficacy of four DNA markers and their combinations (rbcL, matK, ITS, trnH-psbA) as barcode markers were tested across the endangered Paphiopedilum species from Peninsular Malaysia. Four species of Paphiopedilum were sampled and barcoded. The DNA barcodes reliabilities were evaluated using NCBI BLASTn program, phylogenetic tree via Neighbour-Joining method with 1000 bootstrap replicates in MEGA 6 and barcoding gap assessment. matK is the most promising barcode with high sequence quality (100%), high accuracy in BLASTn (100%), clear resolution of species in Neighbour-Joining phylogenetic tree (100%) and a distinct barcoding gap followed by ITS, trnH-psbA and rbcL. The com- bination of barcode regions revealed the lack of variation in rbcL and trnH-psbA but they are still useful for preliminary identification followed up by matK for accurate identification. Key Words: DNA barcodes, Paphiopedilum, identification, endangered species, taxonomy Introduction Peninsular Malaysia has been blessed with a large number of plant species with high representation of the Orchidaceae family including the genus Paphiopedilum.
    [Show full text]
  • ORCHIDACEAE) by Gilbert D
    POLLEN MORPHOLOGY OF THE CYPRIPEDIOIDEAE AND THE APOSTASIOIDEAE (ORCHIDACEAE) by Gilbert D. Newton * and Norris H. Williams* INTRODUCTION Various taxonomic systems have been proposed for the Orchidaceae. Dressler and Dodson (1960) divided the Orchidaceae into two subfamilies: Cypripedioideae and Orchidoideae. The Orchidoideae is the larger subfamily and they considered it the more advanced because the androecium is reduced to one anther. In contrast, the Cypripedioideae contains species with either two or three anthers. Dressler and Dodson considered the Apostasieae and Cypripedieae as tribes in the subfamily Cypripedioideae, but maintained that the Apostasieae may not be related to the Cypripedieae even though they show the same basic plan of flower structure. The Apostasieae have unspe­ cialized four-locular anthers with powdery pollen, the most primitive pollen­ unit condition in the Orchidaceae. In the Cypripedieae the grains are not united into larger units, but the pollen is rather viscid, a condition also con­ sidered primitive. Dressler (1974) later classified both groups as subfamilies, Apostasioideae and Cypripedioideae, and described them as relict groups, each with a few living genera. Garay (1972) disagrees with Dressler and Dodson (1960) that the Apos­ tasieae is a tribe in the Cypripedioideae. According to Garay (1972), the only two genera in the group, Apostasia and Neuwiedia, are not even closely related, but are relict survivors of ancient anagenetic lines. Yet Rao (1974) stated that the external morphology of the flowers and their vasculature show a close affinity between the two genera. Nevertheless, Garay (1972) and Dressler (1974) maintain that the Apostasioideae should be included in the Orchidaceae if the Cypripedioideae are included.
    [Show full text]
  • Network Scan Data
    SelbY!ln!l 7: 129-247 THE RELATIONSHIPS OF THE SLIPPER ORCHIDS (SUBFAMILY CYPRIPEDIOIDEAE, ORCHIDACEAE) John T. A twood, Jr. * ABSTRACT The phylogenetic relationships of the Cypripedioideae are adumbrated by the Wagner Groundplan-divergence method. The mutual resemblance be­ tween paleotropical Paphiopedilum and neotropicalPhragmipedium is doubt­ fully superficial since both share several derived vegetative character states and similar floral bud development. For these genera a simplified classifi­ cation is proposed, and one new taxonomic combination is provided. Selenipedium grades into Cypripedium via C. irapeanum and C. caUtor­ nicum. The correlation of reduced vegetative features with increasing lati­ tude suggests that temperate climates have altered the northern taxa most. By eliminating these features from cladistic analysis, C. arietinum (for which the genus Criosanthes is recommended) is parsimoniously distinct from the 3e/enipedium-Cypripedium clade. Similar flowers of Phragmipedium schlimii and Paphiopedilum subgenus Brachypetalum relate to neoteny, since the floral parts resemble those of young flower buds of related species. Their superficial floral similarity is in­ terpreted as a convergence. Chromosome numbers are reported for five species of Phragmipedium and nine species of Paphiopedilum. Since the basal chromosome number is interpreted as 2n = 20 for Cypripedium, Criosanthes, and Phragmipedium, this is probably the basal number for the subfamily. Although centric fission accounts for the upward aneuploid series in Paphiopedilum (2n = 26-44), higher metacentric arm ratios in the aneuploids than in the non-aneuploids suggest that centric fusion has occurred as well. It is argued that both fission and fusion have karyologically repatterned Paphiopedilum section Barbata with concurrent adaptation to florest floors.
    [Show full text]