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 In Vitro Propagation and Cryopreservation of Lady’s Slipper Orchid Thesis Title (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. ………………………….Signature (Mr. Sutthinut Soonthornkalump) Candidate v ชื่อวิทยานิพนธ์ ผู้เขียน สาขาวิชา ปีการศึกษา การขยายพันธุ์ในสภาพหลอดทดลองและการเก็บรักษาพันธุกรรมในสภาพต่า กว่าจุดเยือกแข็งของรองเท้านารีขาวสตูล (Paphiopedilum niveum (Rchb.f.) Stein) นายศุทธิณัฏฐ์ สุนทรกลัมพ์ ชีววิทยา 2561 บทคัดย่อ รองเท้านารีขาวสตูล (Paphiopedilum niveum (Rchb.f.) Stein) เป็นกล้วยไม้หา ยากที พบได้เฉพาะพื้นทีภาคใต้ของประเทศไทยและทางตอนเหนือของประเทศมาเลเซีย ปัจจุบัน ประชากรในธรรมชาติมีแนวโน้มลดลงจากการลดลงของพื้นทีป่าและการลักลอบเก็บเพือการค้าแบบ ผิดกฎหมาย ท่าให้รองเท้านารีขาวสตูลถูกจัดเป็นพืชใกล้สูญพันธุ์ตามบัญชี 1 ของอนุสัญญาว่าด้วย การค้ าระหว่างประเทศ ซึ งชนิด สัตว์ ป่าและพื ชป่ าที ใกล้จ ะสูญ พั น ธุ์ (CITES) และเนื องจากการ ขยายพันธุ์ด้วยวิธีธรรมชาติและวิธีดั้งเดิมใช้ระยะเวลายาวนานและได้ต้นพันธุ์จ่านวนน้อย จึงท่าให้การ พัฒนาวิธีการขยายพันธุ์ในสภาพหลอดทดลองและการเก็บรักษาพันธุกรรมในสภาพต่ากว่าจุดเยือก แข็งมีความจ่าเป็นต่อการอนุรักษ์พันธุกรรมของรองเท้านารีขาวสตูลนอกสภาพถินอาศัยในระยะยาว การศึกษานี้ประกอบด้วย 1) การศึกษาผลของสารควบคุมการเจริญเติบโตต่อการขยายพันธุ์รองเท้า นารีขาวสตูลด้วยการชักน่าและการเพิมจ่านวนโพรโทคอร์มไลค์บอดี้หรือโซมาติก เอ็มบริโอจากโพรโท คอร์ม และ 2) การเก็ บ รัก ษาพั นธุ ก รรมในสภาพต่า กว่าจุด เยือ กแข็ง ด้ วยวิธี V cryo-plate จาก การศึกษาการขยายพันธุ์รองเท้านารีขาวสตูลพบว่าโพรโทคอร์มอายุ 4 เดือนทีได้จากการเพาะเมล็ด สามารถชักน่าให้เกิดโซมาติกเอ็มบริโอได้โดยตรง เมือเพาะเลี้ยงบนอาหารสูตร Modified Vacin and Went (MVW) ทีมี 1-Naphthaleneacetic acid (NAA) ความเข้มข้น 0.1 มก./ล. ในสภาวะไม่มีแสง เป็นเวลา 3 เดือน และโซมาติกเอ็มบริโอรุ่นที 1 สามารถเพิมจ่านวนเป็นโซมาติกเอ็มบริโอรุ่นที 2 เมือ เพาะเลี้ยงบนอาหารสูตร MVW ทีไม่มีสารควบคุมการเจริญ เติบโตของพืชเป็นระยะเวลา 2 เดือ น สภาวะมีแสง และสามารถชักน่าโซมาติกเอ็มบริโอให้เป็นต้นอ่อนเมือเพาะเลี้ยงบนอาหารสูตรชักน่า ต้นอ่อนเป็นระยะเวลา 4 เดือน และต้นอ่อนทีเกิดจากโซมาติกเอ็มบริโอรุ่นที 2 (V2) และ 3 (V3) มี รูปแบบพันธุกรรมไม่แตกต่างจากต้นอ่อนทีเกิดจากโพรโทคอร์มเริมต้น (V1) ส่าหรับวิธีการทีเหมาะสม ในการเก็บรักษาโซมาติกเอ็มบริโอในสภาพต่ากว่าจุดเยือกแข็งด้วยวิธี V cryo-plate พบว่าโซมาติก เอ็มบริโอ (ขนาดเส้นผ่านศูนย์กลาง 1-1.5 มม) สามารถมีชีวิตรอดได้ (20%) ภายหลังการเก็บรักษาใน ไนโตรเจนเหลว เมือผ่านการ Precondition โดยเพาะเลี้ยงบนอาหารสูตร MVW ทีมีน้่าตาลซูโครส 0.1 M เป็นเวลา 7 วัน ตามด้วยการ Preculture บนอาหารสูตร MVW ทีมีน้่าตาลซูโครส 0.2 M และ 0.6 M เป็นเวลา 1 วัน ตามล่ าดับ แล้ว น่าโซมาติ ก เอ็ ม บริโ อวางติดลงบนแผ่น cryo-plate ด้ว ย alginate gel แล้วแช่ในสารละลาย loading solution (LS) ทีมีกลีเซอรอลเข้มข้น 2 M และ น้่าตาล ซูโครสความเข้มข้น 1.2 M เป็นเวลา 30 นาที จากนั้นย้าย cryo-plate ทีมีโซมาติกเอ็มบริโอติดอยู่ลง vi แช่ ใ นสารละลาย Plant Vitrification Solution 2 (PVS2) เป็ น เวลา 60 นาที และการเติ ม กรด แอสคอร์ บิ ก (Ascorbic acid) ความเข้ ม ข้ น 0.1 mM ในอาหารเพาะเลี้ ย งในวั น ที 7 ของการ Precondition สามารถลดปริมาณอนุมูลอิสระรวม (Total Reactive Oxygen Species, ROS) และ สาร Malondialdehyde (MDA) ในโซมาติกเอ็มบริโอและช่วยเพิมอัตราการรอดชีวิตเป็น 39% อย่าง มีนัยส่าคัญทางสถิติ vii Author In Vitro Propagation and Cryopreservation of Lady’s Slipper Orchid (Paphiopedilum niveum (Rchb.f.) Stein) Mr. Sutthinut Soonthornkalump Major Program Biology Academic Year 2018 Thesis Title ABSTRACT The snow white lady slipper orchid (Paphiopedilum niveum (Rchb.f.) Stein) is an endangered species that distributed in Southern Thailand and Northern Malaysia. The habitat destruction and over-collecting decreased the natural population. Even though P. niveum was protected by Appendix I of the CITES but the conventional propagation method provided low productivity and also take a long period of time which could not meet the commercial demand. Thus, the conservation of genetic resources of P. niveum has required the micropropagation and cryopreservation for long-term storage. This study was composed of 1) the study of the effect of plant growth regulators (PGRs) on the direct somatic embryogenesis and SEs proliferation from protocorm and 2) cryopreservation via V cryo-plate method with the application of ascorbic acid (AA). The results showed that SEs could be generated from four-month-old protocorm when culturing on modified Vacin and Went (MVW) containing 0.1 mgl-1 1Naphthaleneacetic acids (NAA) for 3 months under the dark condition. These primary SEs could be proliferated into secondary SEs after when culturing on free-PGRs MVW in light condition and continuously developed into plantlets after being transferred to culture on plantlet induction medium for 4 months. The uniformity of the genetic pattern between the mother plant (V1) and regenerated plants (V2 and V3) was evaluated by RAPD analysis. The results of cryopreservation using V cryo-plate method showed that the cryopreserved P. niveum SEs could survive (~20%) after precondition on MVW containing 0.1 M sucrose (7 days) followed by two-step preculture on MVW containing 0.2 M and 0.6 M sucrose (each with 1 day). SEs were embedded on cryo-plate with alginate gel before put into loading solution containing 2 M glycerol and 1.2 M sucrose for 30 min. The SEs was dehydrated with plant vitrification solution 2 (PVS2) for 60 min. The application of ascorbic acid (AA) in the critical step could reduce total reactive oxygen species (ROS) and malondialdehyde (MDA) production which significantly improved the survival percentage to 39% of cryopreserved P. niveum SEs. viii ACKNOWLEDGMENTS I would like to express my special appreciation to my advisor, Assoc. Prof. Dr. Upatham Meesawat, for providing informative advices and supporting throughout my thesis. This thesis cannot be accomplished without her patient proofreading towards the completion. I am grateful to Asst. Prof. Dr. Korakot Nakkanong from Department of Plant Science, Faculty of Natural Resources, Prince of Songkla University (PSU), for her valuable guidance and supports, especially in techniques of molecular biology. I would also like to extend my sincere thanks to Dr. Shin-ichi Yamamoto for his kindly helps and a good opportunity in our collaboration. I have learned a lot of cryopreservation techniques under his kind supervision. He also gave the wonderful experience when I stayed in Genetic Resources Center, The National Agriculture and Food Research Organization (NARO), Tsukuba, Japan. My gratefulness are also due to Dr. Takao Niino and the members of cryopreservation laboratory, NIAS, Tsukuba, Japan, for their warm welcome and supporting me. I would also like to thank Assoc. Prof. Dr. Kanchit Thammasiri, Department of Plant Science, Faculty of Science, Mahidol University (MU) and Prof. Dr. Sompong Te-chato, Department of Plant Science, Faculty of Natural Resources, PSU for serving as my committee members and for their brilliant comments and criticism. Moreover, I would like to thanks Asst. Prof. Dr. Ngarmnij Chuenboonnagarm and Asst. Prof. Dr.Thaya Jenjittikul who always encourages and supports me in the difficult times through my Ph.D. study. I would also like to express thanks to all members Plant Biotechnology Research Unit and Plant Physiology Research Unit members from Department of Biology, Faculty of Science, PSU, especially Asst. Dr. Pimchanok Buapet for all their helps and valuable suggestions through my research. I would like to thank Dr. Natthakorn Woraathasin and members of the molecular genetic laboratory from the Department of Plant Science, Faculty of Natural resources, PSU for technical support. As well as, I am very grateful to Ms Nopparat Thawinwathin from the Department of Agriculture Extension, as well as Mr. Ongart Tantawanich, and Mr. Mohammat Nueaoon contributing capsules of P. niveum for my study. I wish to express my gratitude to Ms Lily Chen for her untiring help in the first manuscript preparation. ix This research was contributed financial by Graduate School, PSU for providing Graduate Scholarship grant (95000201), research funding and scholarship for support exchange students and international credit transferred through ASEAN Community. Finally, I would acknowledge with special gratitude to my beloved parent, my sister, my family and Ms Kanokwan Buakeeree for their selfless love for spending difficult time with me and giving worthy encouragement. Sutthinut Soonthornkalump x CONTENT Page บทคัดย่อ v ABSTRACT vii ACKNOWLEDGMENTS viii CONTENTS x LIST OF TABLES xiii LIST OF FIGURES xiv LIST OF ABBREVIATIONS xv CHAPTER 1 INTRODUCTION 1.1 Introduction 1 1.2 Review of literature 2 1.2.1 The genus Paphiopedilum and subgenus Brachypetalum 2 1.2.2 Paphiopedilum niveum (Rchb.f.) Stein 2 1.2.2.1 Distribution and botanical description 2 1.2.2.2 Conservation situation and ex situ conservation 7 1.2.3 Micropropagation and somatic embryogenesis of Paphiopedilum 7 1.2.4 Cryopreservation based on vitrification and V cryo-plate technique 11 1.2.5 The uses of ascorbic acid as antioxidant in cryopreservation 16 1.2.6 Genetic alteration and molecular marker detection by RAPD 16 1.3 Objectives 19 CHAPTER 2 RESEARCH METHODS 2.1 Plant materials and culture medium 20 2.2 Seed pretreatment 20 2.3 Somatic embryo (SEs) induction, proliferation assessment 22 2.3.1 SEs induction 22 2.3.2 SEs proliferation 23 2.2.3 Plantlet regeneration 23 2.3.4 Histological observation 24 2.4 RAPD assessment 24 2.4.1 Genomic DNA extraction 24 2.4.2 Primer screening, RAPD amplification, and analysis 24 xi CONTENT (CONTINUED) Page 2.5 V cryo-plate protocol, total ROS and MDA determination 26 2.5.1 Two-step preculture 26 2.5.2 SE embedding on the cryo-plate 26 2.5.3 Osmoprotection and dehydration 27 2.5.4 Cryopreservation in liquid nitrogen, thawing and regrowth 27 2.5.5 Water content determination 29 2.5.6 Ascorbic acid supplementation in the optimized V cryo-plate 29 method 2.5.7 Determination of total ROS 29 2.5.8 MDA analysis 29 2.6 Statistical analysis CHAPTER 3 RESULTS 3.1 In vitro cloning via direct somatic embryogenesis 30 31 30 3.1.1 SEs induction 31 3.1.2 SEs proliferation 33 3.1.3 Developmental pattern via direct somatic embryogenesis 34 3.1.5 Genetic variation assessment using RAPD 37 3.2 Cryopreservation by V cryo-plate method 3.2.1 The optimization of the V cryo-plate method and water content 39 39 determination 3.2.2 Effect of ascorbic acid supplementation on total ROS, MDA level 42 and survival of AA treated-SEs during the optimized V cryo-plate method CHAPTER 4 DISSCUSSION 4.1 In vitro cloning by direct somatic embryogenesis 47 47 4.1.1 SEs induction 47 4.1.2 SEs proliferation 48 4.1.3 Histological observation 49 4.1.4 RAPD analysis 49 4.2 Cryopreservation by V cryo-plate method 50 xii CONTENT (CONTINUED) Page CHAPTER 5 CONCLUSIONS 52 REFERENCES 53 APPENDICES 65 VITAE 86 xiii LISTS OF TABLES Tables Table 1 Description of morphological characteristics of Paphiopedilum niveum Table 2 Previous reports on somatic embryogenesis induction in Page 5 9 Paphiopedilum spp. Table 3 Summarized preculture conditions of the vitrification-based protocol 13 Table 4 Summarized osmoprotectant treatment of the vitrification-based 14 protocol Table 5 Summarized incubation time for dehydration using PSV2 15 Table 6 Techniques in molecular marker 17 Table 7 Detection of somaclonal variation-derived of in vitro culture using 18 various molecular marker techniques Table 8 RAPD primers used for the genetic fidelity assessment of in vitro 25 Paphiopedilum niveum among the mother (V1) and the regenerant plants (V2-V3) Table 9 Effects of NAA and TDZ on SE induction of Paphiopedilum niveum. 32 Data derived from four-month-old protocorms cultured on MVW medium supplemented with various concentrations of PGRs and grown in darkness for 3 months. Table 10 Effects of NAA and kinetin on SE proliferation, survival, and color of 33 the regenerated SEs of Paphiopedilum niveum. Table 11 Factors (2nd preculture, osmoprotection and dehydration) affecting the 40 survival percentage of Paphiopedilum niveum SEs. Table 12 Three-way ANOVA with interaction between all combinations of three main factors (preculture; P, osmoprotection; O and dehydration; D) exhibiting in cryopreserved Paphiopedilum niveum SEs via V cryoplate method 41 xiv LISTS OF FIGURES Figures Figure 1 Biodiversity and distribution of subgenus Brachypetalum of the Page 4 world Figure 2 Morphology of P. niveum 6 Figure 3 Overview of methodology of this study 20 Figure 4 Schematic diagram of plant material preparation and the experiment 21 of Paphiopedilum niveum micropropagation Figure 5 Summarized procedure of direct SEs induction 22 Figure 6 Summarized diagram of SEs proliferation and plantlet regeneration 23 Figure 7 Summarized diagram of the cryopreservation optimization by V 28 cryo-plate Figure 8 Morphological characteristics during SE formation and proliferation 35 of Paphiopedilum niveum. Figure 9 Histological observation on direct somatic embryogenesis (DSE) of 36 Paphiopedilum niveum. Figure 10 Gel electrophoresis of RAPD from clone 1–3 of Paphiopedilum 37 niveum showing monomorphic banding patterns generated using ten selected primers. Figure 11 Diagrammatic summary of in vitro cloning of genetically uniform 38 Paphiopedilum niveum via direct somatic embryogenesis (DSE) Figure 12 Percentage of water content in Paphiopedilum niveum SEs during 42 cryopreservation using V cryo-plate method Figure 13 Determination of total ROS and MDA level during the optimized V 44 cryo-plate method of non AA-treated and AA-treated cryopreserved Paphiopedilum niveum SEs Figure 14 Survival percentage of non AA-treated and AA-treated 45 Paphiopedilum niveum SEs compared to non-cryopreserved control SEs. Figure 15 Schematic diagram of cryopreservation of Paphiopedilum niveum SEs using V cryo-plate method 46 xv LISTS OF ABBREVIATION AA ABA AC ANOVA AFLP BA BH CAPS CITES CRD CW D DCFDA DMRT DMSO DNA DSE DV 2,4-D EV GS H ISSR LS LN MDA MS MVW NAA NIAS PCR PLBs PGRs PSU PUFA PVS2 PVP-40 RAPD RFLP ROS SCAR SCoT SE SSR SV TBA Ascorbic acid Abscisic acid Activated charcoal Analysis of variance Amplified Fragment Length Polymorphism 6-Benzylaminopurine Banana homogenate Cleaved Amplified Polymorphic Sequence Convention on International Trade in Endangered Species of Wild Fauna and Flora Completely randomized design Coconut water Day 2,7-Dichlorodihydrofluorescein diacetate Duncan multiple range test Dimethyl sulfoxide Deoxyribonucleic acid Direct somatic embryogenesis Droplet vitrification 2,4-Dichlorophenoxyacetic acid Encapsulation vitrification Graduate school Hour Inter Simple Sequence Repeat Loading solution Liquid nitrogen Malondialdehyde Murashige and Skoog medium Modified Vacin and Went medium 1-Naphthaleneacetic acid National Institute of Agrobiological Sciences Polymerase chain reaction Protocorm-like bodies Plant growth regulators Prince of Songkhla University Polyunsaturated fatty acid Plant vitrification solution 2 Polyvinylpyrrolidone Random Amplified of Polymorphic DNA Restriction Fragment Length Polymorphism Reactive oxygen species Sequence Characterized Amplified Region Start Codon Targeted Somatic embryo Simple Sequence Repeat Somaclonal variation Thiobarbituric acid xvi TCA TDZ V VC VW Trichloroacetic acid Thidiazuron Vitrification V cryo-plate Vacin and Went medium 1 CHAPTER 1 INTRODUCTION 1.1 Introduction Paphiopedilum niveum (Rchb.f.) Stein is an endangered orchid which distributed in Southern Thailand and Northern Peninsular Malaysia (Pedersen et al., 2011). Its conservation status is a serious concern due to climate change, increases fragmentation of wild population from the decreasing of habitat (Seaton et al., 2010). Moreover, the increase of harmfully illegal collection pressured on natural populations of Paphiopedilum (Zeng et al., 2013). Thus, the efficient conservation procedure is an urgent requirement. In term of conservation, simultaneously of mass clonal propagation and long-term storage of germplasm of P. niveum using cryopreservation may achieve the goal of P. niveum conservation. Plant tissue culture seems to be the suitable resolution to reduce the pressure of wild population of P. niveum. Because it was employed in mass production in short time and reasonable cost (Liao et al., 2011). Many previous experiments revealed that the application of single or a combination of classical plant growth regulators (PGRs) can affect plant multiplication. However, the use of PGRs may be a cause of genetic alteration in various types such as an alteration of cytological and chromosomal mutation, leading to somaclonal variation (Samarfard et al., 2013). The occurrence of somaclonal variation has been defined as a serious problem in the conservation process (Sarasan et al., 2006). There are many detection methods of somaclonal variation such as morphological observation, cytological and molecular techniques (Rao, 2004). The simplest and reasonable molecular method is Random amplification polymorphism DNA (RAPD) which very useful in the clonal fidelity assessment (Rao 2004). The previous study used RAPD to assess the genetic homogeneity of regenerated plantlets from seedlings of Aerides vandarum x Vanda stangeana cultured on a multiple shoot induction medium (½MS + 2 mgl-1 TDZ) (Kishor and Devi, 2009). While the genotypic alteration at 5.81% was found in the regenerant PLBs induced from in vitro pseudostem of Cymbidium giganteum which was detected the genotypic alteration by RAPD after cultured on MS contained 0.909 µM TDZ (Roy et al., 2012). It demonstrated the probability of somaclonal variation is linked to the type of explant, type of PGRs and PGR concentration. V cryo-plate, a novel technique of cryopreservation, is the application of vitrification and the use of a small aluminum plate with wells. The V cryo-plate provided rapid thermal exchange rate which enables to reduce the damage from ice crystal formation (Vujović et al., 2011). Moreover, the use of V cryo-plate is easy to handle many pieces of explant in the process of cryopreservation and can reduce the loss of plant material (Yamamoto et al., 2011A). The V 2 cryo-plate technique has been investigated in economic crops such as mulberry (Morus sp.) (Yamamoto et al., 2011A), mint (Mentha spp.) (Yamamoto et al., 2012A), Dalmatian chrysanthemum (Tanacetrum cinerariifolium) (Yamamoto et al., 2011B), strawberry (Fragaria x ananassa) (Yamamoto et al., 2012B) and carnation (Dianthus caryophyllus) (Sekizawa et al., 2012). Recently, the literature review showed the limitation of orchids cryopreservation especially, Paphiopedilum species. Moreover, the use of V cryo-plate method in orchids cryopreservation is taken in the initiation step. Thus, this study focused on the investigation of PGRs induction of direct somatic embryogenesis, the proliferation of somatic embryo, histological observation and genetic stability assessment using RAPD. The optimized protocol of V cryo-plate method and the effect of ascorbic acid as an antioxidant to improve the survival rate of post-cryopreserved P. niveum PLBs were also determined. 1.2 Review of Literature 1.2.1 The genus Paphiopedilum and subgenus Brachypetalum Paphiopedilum is a small to large orchids with the deeply slipper-shaped labellum which classified to subfamily Cypripedioideae of the family Orchidaceae (Pedersen et al., 2011). There are 3 subgenera were classified to this genus, Brachypetalum, Pahiopedilum and Megastaminodium. The subgenus Brachypetalum was comprised 3 sections, Pavisepalum (6 sp.), Concoloria (6 sp.) and Emersoniana (2 sp.) (Cribb, 2014; Górniak et al., 2014; Koopowitz et al., 2017). There are 14 species from subgenus Brachypetalum (Concoloria 5 sp.) and Pahiopedilum (Pardalopetalum 1 sp.; Pahiopedilum 5 sp. and Barbata 3 sp.) distributed in Thailand (Pedersen et al., 2011). The section of Concoloria was comprised of 6 sp. with 6 varieties (Fig. 1) distinguished by their markedly tessellated adaxial and densely maroon spotted at abaxial of leaves. The flower color is pale yellow-cream or white with scattered purple dot and tridentate apex of staminode (Cribb, 2014; Averyanov et al., 2017; Koopowitz et al., 2017). The center of distribution area of Concoloria is Thailand and Indochina, 5 species with 3 varieties were found in almost part of Thailand which composed of P. bellatulum, P. concolor, P. niveum, P. thaianum and 3 varieties of P. godefroyae (Fig. 1) (Pedersen et al., 2011; Cribb, 2014). 1.2.2 Paphiopedilum niveum (Rchb.f.) Stein 1.2.2.1 Distribution and botanical description Paphiopedilum niveum is distributed in the shaded area of limestone of Southern Thailand to Northern Peninsular Malaysia at 0-200 m above sea level (Pedersen et al., 2011). The flowering season is April-July (summer) but also possible flowering in all year 3 round (Braem and Öhlund, 2016). According to Pedersen et al. (2011), the descriptive data of P. niveum characteristics and botanical description were shown (Fig. 2 and Table 1). Figure 1 Biodiversity and distribution of subgenus Brachypetalum of the world (constructed from Pedersen et al., 2011; Cribb, 2014; Averyanov et al., 2017; Koopowitz et al., 2017) *endemic to Thailand 4 Table 1 Description of morphological characteristics of Paphiopedilum niveum (adopted from Pedersen et al., 2011) Plant part Leaves Quantity 4-5 Peduncle Bracts 1 1 Flowers 1-3 Length (cm) Width (cm) 8-19 2.4-3.6 >20 1.1-1.4 1-1.2 - Ø 6-8 Shape Color Narrowly, elliptic, rounded and minutely emarginated apex, ciliate at the base Broadly ovate, obtuse Mottled very dark and pale green above, heavily dotted purple below - Purple with shortly dense white pubescent White to pale green, spotted purple White often highly dotted purple towards base of segment and front of labellum. Pubescent on outside and at base of petals White often highly dotted purple towards base. Dorsal sepal 1 2.7-4.2 3-5 Very broadly ovate, obtuse to emarginate Synsepal 1 2-3 1.5-3 Concave, ovate, obtuse White often highly dotted purple towards base. Lateral petal Labellum 2 3.3-4.3 2.2-3.9 Elliptic, rounded with shortly ciliate on margins White often highly dotted purple towards base. 1 2.2-3.6 1.5-1.8 Ovoid to ellipsoidal with incurve margins White often highly dotted purple towards base. Column 1 - - White Ovary 1 4.5 - Sessile or shortly stalked, glabrous Stigma stalked, tripartite less papillose Inferior, long with densely shortly pubescent Staminode 1 0.6-0.9 1-1.2 White with yellow blotched at the center Anther (pollinia) 2 0.15 Broader than long, transversely subelliptic, one-to three-toothed at apex Bilocular, borne on short filament 0.15 Green flushed with dark purple Yellow, pollen viscid 5 6 Figure 2 Morphology of P. niveum: A flowering plant; B Front view of flower showing dorsal sepal (D), lateral petal (P), labellum (L) and staminode (St); C Rear view of flower: synsepal (Sp), ovary (O), D Capsule (C), bract (B); and peduncle (Pd); E leaf (Lf) 7 1.2.2.2 Conservation situation and ex situ conservation Paphiopedilum niveum has high value and very popular in the worldwide ornamental plant market (Kaewubon et al., 2010). Generally, orchid needs mycorrhizal fungi in root system for exchange essential phosphate and nitrate ions for seed germination and plant development (Athipunyakom et al., 2004). Thus, Paphiopedilum was determined as a slow grower compared with other orchid genera. Due to the situation of the wild orchid was threatened by global climate change and the over-collecting to sell in the domestic market and illegal international trading. Thus, all species of Paphiopedilum were protected by international wild-collected trading by listed in Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix I (McGough et al., 2006). CITES Appendix I is the most strictly prohibited wild-collected living or dead specimens. However, trading of plant or plant part from artificial propagation is allowed with permission which controlled by particularly strict regulation of CITES (CITES, 2014). Recently, the conventional method of Paphiopedilum propagation has unreliable to fulfill commercial demand due to low productivity and extremely time-consuming (Ng et al. 2010; Ng and Saleh 2011). For instance, the encouraging of artificial cultivation by in vitro culture can facilitate massive propagation leading to the reduced wild-collected pressure of medicinal orchid such as Dendrobium catenatum (Liu et al., 2014). The previous study reported that the transplanted seedling of Paphiopedilum wardii demonstrated the survivability in natural habitat in Yunnan and Guangdong at 49.67-60.33% after reintroduced for 2 years (Zeng et al., 2012). So the micropropagation of Paphiopedilum would be the efficient tool to supply the artificially propagated plant to the orchid collector and reduce wild-collecting pressure from the natural population. 1.2.3 Micropropagation and somatic embryogenesis of Paphiopedilum The conventional propagation by bud division is unreliable and cannot meet market demand because of the extreme time-consuming (Huang et al., 2001). So, mass propagation of Paphiopedilum may reduce the pressure of wild population. Micropropagation is an effective tool to produce large-scale clonal propagation in many plants including orchids (Martin and Madassery, 2005). Since biotechnology was applied in plant tissue culture, somatic embryogenesis has become ideally method for large-scale cloning of valuable cultivar (Deo et al., 2010). Large scale propagation via somatic embryogenesis is an efficient method which could be facilitated in many branches of plant biotechnology such as, massive clonal propagation, artificial seed production, gene transformation and also been an effective model in the molecular and morphogenetic event (Deo et al., 2010). The somatic embryogenesis is the 8 dedifferentiation of a somatic cell to somatic embryo (SE) without vascular tissue which connected to parental tissue (Jiménez, 2005). Orchid SE, known as protocorm like-bodies (PLBs), has special characters from the other higher plants cause of the distinctive morphology (Lee et al., 2013). There are two regeneration pathways of somatic embryogenesis; direct somatic embryogenesis and indirect somatic embryogenesis. Direct somatic embryogenesis is the direct regeneration of SE from explant without callus formation (Chawla, 2000) while indirect somatic embryogenesis is the regeneration that intervening callus phase and subsequently forms the somatic embryo (Ji et al., 2011). Generally, the somatic embryogenesis induction composed of two stages including induction and expression. The proper modification of culture condition (medium and PGRs) could stimulate the reversibility of competence somatic cell (Jiménez, 2005). Expression stage might be activated by reduction of PGRs, particularly auxin (Jiménez, 2001). Auxin is known as a promoter of callus proliferation and an inhibition of cell differentiation. While cytokinin plays an important role in embryogenic cell formation and stimulates maturation of somatic embryo (Chawla, 2000; Deo et al., 2010). Supplementation of auxin and cytokinin in SE induction were presented in several studies which were summarized in Table 2. It demonstrated that the most popular auxin and cytokinin were 1-naphthaleneacetic acid (NAA) and thidiazuron (TDZ), respectively. Table 2 Previous reports on somatic embryogenesis in Paphiopedilum spp. References - 5 mgl-1 2,4-D + 1 mgl-1 TDZ - - Lin et al, 2000 5 mgl-1 2,4-D + 1 mgl-1 TDZ in darkness 5 mgl-1 2,4-D + 1 mgl-1 TDZ in darkness 0.5 mgl-1 NAA + 0.1 mgl-1 TDZ - Hong et al, 2008 1 mgl-1 2,4-D + 0.1 mgl-1 TDZ in darkness - 0.1 mgl-1 NAA + 0.5 mgl-1 TDZ + 1% sucrose - Kaewubon et al., 2010 0.86 mgl-1 kinetin - 0.86 mgl-1 kinetin Free-PGRs 5 mgl-1 2,4-D + 1 mgl-1 TDZ - 5 mgl-1 kinetin + 2 mgl-1 BA Ng & Saleh, 2011 Zeng et al., 2013 Explant Basal medium Callus induction P. callosum x lawrenceanum Protocorm P. Alma Gavaert Seedderived callus P. niveum Seedderived callus P. rothschildianum Stem nodal ½ strength MS + 100 mg/l myoinositol + 0.5 mgl-1 nicotinic acid + 0.5 mgl-1 pyridoxine HCl + 0.1 mgl-1 thiamine + 2 mgl-1 glycine + 1 gl-1 peptone + 170 mgl-1 NaH2PO4 + 2% sucrose + 0.22% gelrite ½ MS (1/2 strength macronutrient + full-micronutrient) + 100 mgl-1 myo-inositol + 0.5 mgl-1 nicotinic acid + 0.5 mgl-1 pyridoxine HCl + 0.1 mgl-1 thiamine HCl + 2 mgl-1 glycine + 1 gl-1 peptone + 170 mgl-1 NaH2PO4 + 2% sucrose + 0.22% gelrite MVW (full strength macronutrient VW + ½ strength MS micronutrient) + 100 mgl-1 myo-inositol + 0.5 mgl-1 nicotinic acid + 0.5 mgl-1 pyridoxine HCl + 0.1 mgl-1 thiamine HCl + 2 gl-1 peptone + 2% sucrose + 0.2% gelrite ½ strength MS P. hangianum Protocorm ½ strength MS - 9 PGRs supplemented in basal medium Callus SE SE induction proliferation proliferation Species Table 2 Previous reports on somatic embryogenesis in Paphiopedilum spp. (continued) PGRs supplemented in basal medium Species Explant Basal medium Callus induction Callus proliferation P. niveum Seedderived callus MVW (full strength macronutrient VW + ½ strength MS micronutrient) + 100 mgl-1 myo-inositol + 0.5 mgl-1 nicotinic acid + 0.5 mgl-1 pyridoxine HCl + 0.1 mgl-1 thiamine HCl + 2 gl-1 peptone + 2% sucrose + 0.2% gelrite - 15 gl-1sucrose + 10% Coconut water (CW) SE induction 0.5 mgl-1 2,4-D + 0.1 mgl-1 TDZ SE proliferation References 10 gl-1sucrose + 0.2% AC + 10% CW Chaireok et al., 2015 10 11 1.2.4 Cryopreservation based on vitrification and V cryo-plate technique Cryopreservation is the storage of living materials of plant or animal at an extremely low temperature (lower than -130 °C) and the stored materials can still survive and regrowth after thawing (Day and Stacey, 2007). However, the sufficient dehydration of intracellular water during cryopreservation procedure is essential to investigate (Pegg, 2010). Recently, several cryopreservation procedures have been developed such as controlled rate cooling, dehydration, encapsulation dehydration, vitrification, encapsulation vitrification and dropletvitrification (Kaczmarczyk et al., 2012). Vitrification technique comprised of the step of preculture, osmoprotection, dehydration, rapid warming and regrowth (Sakai and Engelmann, 2007). Preculture is the step which stimulates an endogenous cryoprotectant accumulation in plant cells using high sugar concentration in medium with suitable incubation time (Sakai and Engelmann, 2007). The studies on preculture conditions on the vitrification based protocol were shown in Table 3. After preculture, loading solution (LS) is used as osmoprotectant for protecting plant material from the excessive osmotic stress which could be modified based on a combination of glycerol and sucrose at different concentrations (Table 4). Dehydration using plant vitrification solution 2 (PVS2) is the most common use in this crucial step (Kami, 2012). PVS2 containing glycerol, dimethyl sulfoxide (DMSO), ethylene glycol and sugars in high level has toxic and excessive osmotic stress (Kaczmarczyk et al., 2012). So, the determination of optimal incubation time of PSV2 is needed. The optimal use of PVS2 in previous studies were shown in Table 5. After immersion in liquid nitrogen (LN), rewarming is an essential step to avoid damaging devitrification and prevent ice-formation during rewarming (Sakai and Engelmann, 2007). Cryopreserved tube need to warm in hot water for a few minutes (Kami, 2012) or using 1 M sucrose solution at room temperature for V cryo-plate technique (Sekizawa et al., 2011). The successful of growth recovery after exposure to LN can be determined by green and resumed growth of plant material (Sakai and Engelmann, 2007). Occasionally callus tissue may be regenerated during the regrowth step and possibly use as plant material ( Kaczmarczyk, 2012). However, the risk of occurrence of genetic variation, an undesirable in plant germplasm storage, may be a high frequency (Sakai and Engelmann, 2007). For instance, the isolated Citrus cells from embryogenic callus culture presented the significant change in DNA methylation profile after cryopreservation via vitrification method (Hao et al., 2002). V cryo-plate is one of the most novel technique which has been developed by Japanese scientist team from the National Agriculture and Food Research Organization (NARO) who applied for a small aluminum plate with ten wells on the upper side called V cryo-plate (Yamamoto et al., 2012A). This V cryo-plate has facilitated as plant material holder which 12 reduce the injury and loss of plant material during the cryopreservation process (Yamamoto et al., 2011). The first successful of V cryo-plate method was reported by Yamamoto et al. (2011). This method combines the advantage of vitrification and droplet vitrification (Yamamoto et al., 2011). Therefore, the vitrification has low complexity in the process and no need to use special equipment (Reed, 2008) and the use of aluminum foil of droplet vitrification is facilitated uniformly rapid thermal exchange (Vujović et al., 2011). Moreover, V cryo-plate is useful in transferring of plant material in the steps of LS and PVS2 (Sekizawa et al., 2011). The V cryoplate technique was used in some crop plants such as mulberry (Morus spp.) (Yamamoto et al., 2012C), carnation (Dianthus caryophyllus) (Sekizawa et al., 2011) and several varieties of potato (Solanum tuberosum) (Yamamoto et al., 2012D; Yamamoto et al., 2015). The V cryoplate method was expected to be a new method assisting the ex situ conservation of endangered wild orchids. 13 Table 3 Summarized preculture conditions of the vitrification-based protocol Plant species Explant Doriteanopsis Cell New suspension Method V Preculture time (d) 7 Sucrose (M) References 0.1 +1 mgl-1 Tsukazaki et al., ABA 2000 Toyohashi* Fragaria x Shoot V 1 0.3 Niino et al., 2003 Shoot DV 1 and 2 0.5 Halmagyi and ananassa cv. Donner Rosa spp. Pinker, 2006 Leontopodium Shoot V 1 0.3 2008 hayachinense Brassia rex* Tanaka et al., PLBs V 2 0.5 Shuhaida et al., 2009 Dianthus Shoot VC 2 0.3 2011 caryophyllus Mentha spp. Sekizawa et al., Shoot VC 2 0.3 Yamamoto et al., 2011A Dendrobium PLBs EV 2 0.4 2012 nobile* Anemarrhena Callus V 2 0.5 PLBs V 2 0.4 sonia-28* Solanum niveum* Poobathy et al., 2012 Shoot VC overnight 0.3 Yamamoto et al., 2015 tuberosum Paphiopedilum Hong and Yin, 2012 asphodeloides Dendrobium Mohanty et al., PLBs clump V 5 0.75 Chaireok et al., 2016 V = Vitrification; DV = Droplet vitrification; EV = Encapsulation vitrification; VC= V cryoplate * orchid species 14 Table 4 Summarized osmoprotectant treatment of the vitrification-based protocol Plant species Explant Method Vanilla Shoot DV planifolia* tip Loading Incubation solution time (min) 2 M glycerol + 20-30 0.4 M sucrose References GonzalezArnoa et al., 2008 Dendrobium PLBs EV Shoot VC PLBs EV 90 2 M glycerol + Shoot VC 2 M glycerol + 60 Seed V 30 Seed V PLBs EV Galdiano et al., 2013 10-30 Hu et al., 2013 2 M glycerol + 80 Mohanty et al., 2012 0.4 M sucrose chrysanthum* Solanum 30 0.4 M sucrose formosana* Dendrobium 2 M glycerol + Yamamoto et al., 2012A 0.4 M sucrose flexuosum* Bletilla 2 M glycerol + Mohanty et al., 2012 0.8 M sucrose Oncidium Sekizawa et al., 2011 0.4 M sucrose nobile* Mentha spp. 2 M glycerol + Yin and Hong, 2009 1.4 M sucrose caryophyllus Dendrobium 80 1 M sucrose candidum* Dianthus 2 M glycerol + Shoot VC 2 M glycerol + 30 0.8 M sucrose tuberosum Paphiopedilum PLBs niveum* clump V 2 M glycerol + 0.5 M sucrose Yamamoto et al., 2015 30 Chaireok et al., 2016 V = Vitrification; DV = Droplet vitrification; EV = Encapsulation vitrification; VC= V cryoplate * orchid species 15 Table 5 Summarized incubation time for dehydration using PSV2 Plant species Explant Vanda coerulea* Seed Method Incubation time (min) V 20 References Thammasiri and Soamkul, 2007 Phaius Seed V 60 2009 tankervilleae* Dendrobium PLBs EV 150 Yin and Hong, 2009 candidum* Cymbidium Hirano et al., Seed V 30 and 60 Hirano et al., 2011 finlaysonianum*, C. goeringii* and C. macrorhizon* Vanda Kaseem’s PLBs V 20 Delight* Poobathy et al., 2012 Bletilla formosana* Seed V 30 Hu et al., 2013 Garcinia Shoot V 25 Ibrahim and Normah, 2013 mangostana Paphiopedilum PLBs niveum* clump V 90 Chaireok et al., 2016 V = Vitrification; DV = Droplet vitrification; EV = Encapsulation vitrification; VC = V cryoplate * orchid species 16 1.2.5 The uses of ascorbic acid as an antioxidant in cryopreservation Reactive oxygen species (ROS) is a by-product of the photosynthesis and cellular respiration which was regulated by ascorbate-glutathione cycle which prevents the immoderate reduction or oxidation (Foyer and Noctor, 2011). In general, the high level of ascorbate could be found in the apoplastic channel which acts as a cell membrane defender against ROS (Pereira, 2004). Therefore, the cell membrane is a sensitive organelle which can be damaged by ROS (Shalata and Neumann, 2001). In addition, the damaged polyunsaturated fatty acid (PUFA) from the plasma membrane can generate malondialdehyde (MDA) which can interrupt biological function when it binding with protein (Uchendu et al., 2010). Ascorbic acid (AA) or vitamin C is a well-known antioxidant and very popular use (Arrigoni and De Tullio, 2002). The previous report showed that a few concentration of ascorbic acid (0.005%) was reported to control browning of Cavendish banana (Ko et al., 2009). However, there are a few reports of AA supplementation in plant cryopreservation. The low concentration at 0.5 mgl-1 AA showed the decreased of MDA level leading to improved regrowth rate of blackberry shoot tip when added in any step of vitrification namely, preculture, loading, rinsing, and regrowth (Uchendu et al., 2010). The addition of AA (0.28 mM) in loading solution could improve the survival rate in cryopreserved Nephelium ramboutan-ake shoot tip because AA could decrease the oxidative stress (Chua and Normah, 2011). Hence, these reports revealed the potential of AA as an antioxidant in the reduction of oxidative stress leading to improve the survivability of explant in cryopreservation. 1.2.6 Detection of genetic alteration by molecular marker Somaclonal variation (SV) is any genetic variation types which are detected in cells or tissues cultured in vitro (Evans et al., 1984). This SV may occur permanently or temporarily change in different level such as morphological changes, physiological responses and epigenetic (Bairu et al., 2011). The SV may use as a tool for crop improvement which important in the mutant selection (Lestari, 2006). However, in term of conservation, the uniform of genetic in clonal propagation is an ideal expectation to maintain genetic resources (Bairu et al., 2011). There are many factors affecting SV such as the type of explants, PGRs, culture period, proliferation rate and culture condition lead to a genetic change in the in vitro variants (Skirvin, 1994). Thus the monitoring of genetic stability in plant tissue culture is necessary. The basic molecular marker techniques can divide into 2 categories; polymerase chain reaction (PCR) based technique and non-PCR-based technique (Table 6). In the latter case, this technique detects the DNA polymorphism by the hybridizing digested DNA with the radioactive probe such as the Southern blot. Meanwhile, the PCR-based technique uses the PCR technology and random primers to detect the polymorphism of DNA (Agarwal et al., 2008). Currently, several 17 molecular marker techniques are available as tool to indicate the genetic variation of in vitro cultures (Table 7). For instance, amplified fragment length polymorphism (AFLP) was used to detect the somaclonal variation in the primary regenerant of Echinacea purpurea derived from leaf organogenesis (Chuang et al., 2009). The Random amplification polymorphism DNA (RAPD) was successfully applied to the genetic stability assessment in various clonal micropropagated plants such as Musa spp. (Ray et al., 2006), Clivia miniata (Wang et al., 2012), and Dendrobium nobile (Bhattacharyya et al., 2014). The inter-simple sequence repeat (ISSR) demonstrated the genetic fidelity of regenerant clones from three different explants of Gerbera jamesonii. It was found that the genetic stability was observed in the clone derived from capitulum and shoot tip whereas the SV was found in leaf-derived clones (Bhatia et al., 2009). Table 6 Techniques in molecular marker (adopted from Agarwal et al., 2008) Techniques Type Dominance Abundance RFLP NonPCR Codominant RAPD SSR SCAR ISSR AFLP PCR PCR PCR PCR PCR Dominant Codominant Dominance Dominance Codominant High High Medium Low High High Reproducibility High Low Medium High High High Medium Medium Medium Medium High Medium High Low Low Medium Low-high Medium High Low Low Low Low Medium Degree of polymorphism Technical requirement The quantity of DNA required AFLP: Amplified Fragment Length Polymorphism; ISSR: Inter Simple Sequence Repeat; RAPD: Random Amplified Polymorphism DNA; RFLP: Restriction Fragment Length Polymorphism; SCAR: Sequence Characterized Amplified Region; SSR: Simple Sequence Repeat 18 Table 7 Detection of somaclonal variation-derived of in vitro culture using various molecular marker techniques (adopted from Bairu et al., 2011) Plant species Explant and PGRs Saussurea Callus, NAA and involucrata BA Freesia hybrida Theobroma cacao Molecular marker techniques References RAPD Yuan et al., 2009 Callus, 2,4-D AFLP, MSAP Gao et al., 2010 Embryo, TDZ CAPS Rodríqeuz López et al., 2010 Lilium tsingtauense Embryo, 2,4-D RAPD, ISSR Yang et al., 2010 Cymbidium pseudobulb-derived RAPD, SSR Roy et al., 2012 giganteum PLB, TDZ Dendrobium nobile pseudobulb-derived RAPD, SCoT Bhattacharyya et al., PLB, TDZ 2014 AFLP: Amplified Fragment Length Polymorphism; RAPD: Random Amplified Polymorphism DNA; CAPS: Cleaved Amplified Polymorphic Sequence; ISSR: Inter Simple Sequence Repeat; MSAP: Methylation Sensitive Amplification Polymorphism; SCoT: Start Codon Targeted; SSR: Simple Sequence Repeat 19 1.3 Objectives 1. To examine the effects of auxin (NAA) and cytokinin (TDZ) on somatic embryogenesis and somatic embryo proliferation of P. niveum 2. To determine the genetic fidelity in P. niveum after PGR treatment 3. To optimize the conditions for cryopreservation of P. niveum SEs using the V cryo-plate technique 4. To examine the contents of ROS and MDA during the optimized V cryo-plate protocol after ascorbic acid supplementation 20 CHAPTER 2 RESEARCH METHODOLOGY This study composed 2 main parts which was direct somatic embryogenesis and cryopreservation using V cryo-plate method (Fig. 3) Figure 3 Overview of methodology of this study 2.1 Plant material and culture medium Five-month-old capsule from a hand-pollinated plant of P. niveum was used as plant material. Mother plants were cultured in the shaded greenhouse. The modified Vacin and Went medium (MVW) contained full-strength macronutrient of VW basal medium (Vacin and Went 19 49 ) and half-strength micronutrient of MS (Murashige and Skoog, 19 62 ), 5 mgl-1 of 1000 ppm chitosan (Olizac Technologies, Pathumwan, Bangkok, Thailand), 2 gl-1 BactoTM peptone (Becton, Dickinson and Co., Sparks, MD, USA) and 20 gl-1 sucrose. The pH of medium adjusts to 5.3-5.4 with 1 N NaOH or HCl prior to sterile with autoclave at 121 °C for 20 min. The basal medium for SE induction was solidified with 2 gl-1 phytagelTM (Sigma-Aldrich Co., St. Louis, MO, USA). Addition of 0.72% agar in the medium was used for plantlet regeneration. 2.2 Seed pretreatment The collected capsule was thoroughly washed with tap water and surface-disinfected in 7 0 % ethanol for 3 0 sec and then flame. The flamed capsule was cut longitudinally and the seeds were scooped out and soaked in 1 % ( v/v) clorox® (Clorox Company, Oakland, CA, USA) containing a few drops of Tween 2 0 and occasionally shake for 6 0 min (Shimura and Koda 2 0 0 4 ; Lee, 2 0 0 7 ) . Then, these seeds were rinsed with distilled water for 3 times. Pretreated seeds (approx. 400 seeds) were inoculated into a 125 ml Erlenmeyer flask containing 40 ml of liquid MVW medium. The cultures were placed on agitator at 1 2 0 rpm in darkness at 21 25±2 °C and subcultured at monthly interval for 4 months. Four-month-old protocorm obtained in this step was used as plant material for SEs induction experiment (Fig. 4). Figure 4 Schematic diagram of plant material preparation and the experiment of Paphiopedilum niveum micropropagation 22 2.3 Somatic embryo (SEs) induction, proliferation and genetic fidelity assessment 2.3.1 SEs induction Four-month-old protocorms (approximately 1 - 2 mm in diameter) were cultured on solid MVW supplemented with single and combination of NAA (0 , 0 . 1 , 0 . 3 and 0 . 5 mgl-1 ) ( Fluka Chemie GmbH, Buchs, Switzerland) and TDZ (Sigma-Aldrich Co., St. Louis, MO, USA) (0, 0.1, 0.5 and 1 mgl-1). The cultures were maintained in the darkness and subsequently transferred to fresh medium for 3 months at monthly interval. After 3 months, these treated protocorms were transferred to culture under 1 6 h of photoperiod at the intensity of 2 3 µmol1 m-2s-1 using Philips cool white fluorescent lights at 25±2 °C for a month (Fig. 5). Ten replicate bottles, each with 4 protocorms, were performed for each treatment. The survival percentage (browning protocorm was determined as dead), the percentage of SEs formation and number of SEs per explant was recorded after culture for 3 months. Figure 5 Summarized procedure of direct SEs induction 23 2.3.2 SEs proliferation Protocorm-derived SEs were acclimatized on free hormone solid MVW medium for a month. Then, the SEs clump (approximately 100 mg/clump) were cultured on solid MVW containing NAA (0, 0.1, 0.3 and 0.5 mgl-1) in combination with kinetin (Fluka Chemie GmbH, Buchs, Switzerland) (0, 1, 5 and 10 mgl-1). All treatments, each with 10 replicates (clumps), were subsequently transferred to fresh medium for monthly interval (2 months) and the cultures were maintained under the light conditions as described above. Percentages of the increased fresh weight, rowning, and SE visualization (i.e. vigor and color) were determined after culture for 2 months (Fig. 6). Figure 6 Summarized diagram of SEs proliferation and plantlet regeneration 2.3.3 Plantlet regeneration Regenerated SEs with small leaves induced plantlet regeneration by cultured on solid MVW supplemented with 20 gl-1 sucrose, 2 gl-1 peptone, 2 gl-1 activated charcoal (AC) and 50 gl-1 banana homogenate (BH) for 4 months in light condition as described above. After that, the 24 5 - cm height of vigorous plantlets with well-developed root and shoot were ex vitro and transferred to greenhouse condition. Plantlets were transplanted into 2 inch-nursery-pot containing dried sphagnum moss and growth in the greenhouse at Department of Biology, Faculty of Science, Prince of Songkla University, Hatyai campus, Songkhla, Thailand. 2.3.4 Histological observation Intact protocorm (the control) and regenerant SEs from SEs induction and SEs proliferation were collected and fixed in FAAII (5 : 5 : 9 0 v/v of formaldehyde : glacial acetic acid: 7 0 % ethyl alcohol) for at least 4 8 h. Fixed specimens were dehydrated through tertiarybutyl-alcohol series, infiltrated and embedded in paraplast (Histoplast PE; Richard-Allan Scientific, Kalamazoo, MI, USA). Embedded samples were cut at 6 µm in thickness with a rotary microtome (AO, 820 SPENCER) and affixed to a glass slide. Sections were stained with hematoxylin and safranin (Johansen, 1940) to observe the general structures and to indicate the stage of SE formation. All samples were observed under an Olympus BX 5 1 TRF light microscope (Olympus Optical Co. Ltd., Tokyo, Japan) and photographed by Olympus DP7 2 digital camera (Olympus Optical Co. Ltd., Tokyo, Japan). 2.4 RAPD assessment A comparison of the genetic banding patterns using RAPD between the mother plant (V1 generation) and in vitro regenerated plants (V2 and V3 generation) was performed. 2.4.1 Genomic DNA extraction Fresh leaf tissue (approximately 1 0 mg) was ground to a fine powder in a mortar with liquid nitrogen. Then, DNA was extracted using the DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China) DNA was redissolved with TE buffer (1 0 mM Tris-HCl (pH 8.0) and 1 mM EDTA) and stored at -20 °C in a refrigerator. The concentration and purity of DNA were determined using BioDrop µLITE (BioDrop Ltd., Cambridge, UK) by measuring the absorbance at 2 6 0 / 2 8 0 nm. The quantity test was performed using 1 % agarose gel electrophoresis and automated gel imagining using Gel Doc™ EZ Gel Documentation System (Bio-Rad, California, USA). 2.4.2 Primer screening, RAPD amplification, and analysis A total of 48 primers in operon and UBC series were used for primer screening. Among this primer, ten primers were selected to determine the comparison of genetic pattern among V1 - V3 plants (Table 8). RAPD amplification procedure was followed by Chung et al. (200 6) . A mixture for PCR reaction contained 1 µl of 2 0 ng of template DNA, 2 µl of dNTP (5 mM each of dATP, dGTP, dCTP and dTTP), 1 . 5 µl of 5 0 pM of selected primer, 2 . 5 µl of 1 0 X ThermoPolTM buffer (containing 2 0 mM Tris-HCl, 1 0 mM (NH4 ) 2 SO4 , 1 0 mM KCl, 0 . 1 % Tris®X-1 0 0 , pH 8 . 8 and 2 mM MgSO4 ) , 0 . 2 5 µl (1 unit) of Taq DNA polymerase (New 25 England BioLabs, Massachusetts, USA), and 1 7 . 5 µl of deionized water (2 4 . 5 µl in total volume). The PCR reaction composed of the precycling denaturation at 9 4 °C for 3 min. A cycling protocol was initiated at 94 °C for 40 s, 37 °C for 1 min and 72 °C for 1 min with total cycling at 4 0 cycles. Then the termination of the cycling was done with a final extension at 7 2 C for 10 min using Biometra thermocycler model T-1 (Biometra GmbH, Göttingen, Germany). ° To determine genetic homogeneity, three clones of in vitro plantlets were randomly selected for used in the assessment. The result of DNA banding patterns of each RAPD primer was compared between mother plant (V1 generation) and regenerant plants (V2-V3 generation). Table 8 RAPD primers used for the genetic fidelity assessment of in vitro Paphiopedilum niveum among the mother (V1) and the regenerant plants (V2-V3) Primer Primer nucleotide (5′-3′) Band size Number of scorable Total number of bands/primer/clone bands/primer/clone (bp) Clone1 Clone2 Clone3 Clone1 Clone2 Clone3 OPA-11 CAATCGCCGT 250-2,000 7 4 10 21 12 30 OPA-18 AGGTGACCGT 150-2,200 13 10 11 39 30 33 OPAA-16 GGAACCCACA 250-2,800 6 8 10 18 24 30 OPAB-2 GGAAACCCCT 240-2,000 10 12 10 30 36 30 OPAB-8 GTTACGGACC 320-2,000 10 8 13 30 24 39 OPAD-8 GGCAGGCAAG 300-2,100 14 14 10 42 42 30 OPAD-11 CAATCGGGTC 200-1,900 8 8 11 24 24 33 OPZ-3 CAGCACCGCA 200-1,500 12 7 8 36 21 24 OPZ-11 CTCAGTCGCA 200-2,500 13 9 11 39 27 33 UBC-719 GGTGGTTGGG 200-1,500 9 11 4 27 33 12 102 91 98 306 273 294 Total 26 2.5 V cryo-plate protocol, total ROS and MDA determination This optimized V cryo-plate protocol was partially modified based on the protocol presented by Sekizawa et al. (2011). All treatments employed in this study composed of 3 replicates, each with 10 samples (a single SE) (Fig. 7). 2.5.1 Two-step preculture The SEs (~1-1.5 mm in diameter) were preconditioned on solid MVW with 0.1 M sucrose (7 d) were firstly precultured on the same medium supplemented with 0.2 M sucrose for 1 d (the 1st preculture). The 2nd preculture with the same medium containing different sucrose concentrations (0.4 and 0.6 M sucrose) for a day was tested. 2.5.2 SE embedding on the cryo-plate Precultured SEs, one by one, were placed into wells of aluminum cryo-plate (7 mm x 37 mm x 0.5 mm with Ø 1.5 mm, depth 0.75 mm of ten wells) filled with 2 µl mixture of 2% (w/v) Na-alginate and 0.4 M sucrose in calcium-free liquid MVW. The CaCl2 solution (0.1 M CaCl2 in liquid MVW with 0.4 M sucrose) was added onto cryo-plate and covered completely. After complete polymerization (~15 min at room temperature), the CaCl2 solution was removed by autopipette and the residual solution was absorbed with a piece of filter paper. 27 2.5.3 Osmoprotection and dehydration The cryo-plate with embedded SEs was incubated in the pipetting reservoir containing the amount of 50 ml LS; 2 M glycerol supplemented with various concentrations of sucrose (0.4, 0.8 and 1.2 M) in MVW for 30 min at 25°C. Next, SEs attached to the cryo-plates were dehydrated by placing cryo-plates in the pipetting reservoir filled with 50 ml of PVS2 (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) DMSO and 0.4 M sucrose) for 30, 45 and 60 min at 25°C (Sakai and Engelmann, 2007). 2.5.4 Cryopreservation in liquid nitrogen (LN), thawing and regrowth After that, cryo-plate was put into 2-ml cryotube, uncapped and rapidly plunged into LN for at least 1 h. After thawing in liquid MVW containing 1 M sucrose for 15 min at room temperature, cryo-plate was rinsed with liquid MVW supplemented 2% sucrose. The SE was gently transferred from the alginate, and then the naked SE was cultured on the regrowth medium (Fe-free solid MVW medium containing 0.1 mgl-1 NAA, 0.2% (w/v) PVP-40 and 0.2 % (w/v) AC). These post-cryopreserved SEs were maintained in the darkness for 7 d before being transferred to light condition. The percentage of survival rate and visual observation were evaluated after culture under light condition for 7 d. 28 Figure 7 Summarized diagram of the cryopreservation optimization by V cryo-plate 29 2.5.5 Water content determination (WC) The WC of SEs from the best condition was examined followed by Khoddamzadeh et al. (2011). Three replicates of six SEs from intact SEs (the control), precondition, preculture, osmoprotection and various dehydration period (30, 45 and 60 min) were weighed and then dried in the hot air oven (130 ºC/ 24 h), and then reweighed. The percentage of WC was calculated using the equation below FW = Fresh weight of SEs DW = Dry weight of SEs 2.5.6 Ascorbic acid (AA) supplementation in the optimized V cryo-plate method The AA (0.1 mM) supplementation was conducted on day 7 of culture for 1 d before the beginning of the 1st preculture. The total ROS and MDA content were measured during V cryo-plate method at the 1st preculture, 2nd preculture, osmoprotection and dehydration steps. The intact SE was used as the control. The survival percentage of the AA-treated and the non AA-treated (from both -LN and +LN) was determined. The treatment was performed with 3 replicates, each with 6 samples (SEs). 2.5.7 Determination of total ROS The determining total ROS was measured according to the protocol as described by Jambunathan (2010). The three replications of six SEs were ground in LN. A ground powder of SEs was homogenized with 1 ml of 10 mM Tris buffer (pH 7.2) and centrifuged at 12,000X g for 20 min at 4°C. The 1 ml of sample mixture (100 µl supernatant and 900 µl Tris-buffer) was added with 10 µl of 1 mM DCFDA and then vortexed. The sample mixture was incubated in darkness for 10 min prior to measurement. The sample mixture with DCFDA, the sample mixture without DCFDA (the control) and 1 ml Tris-buffer (the blank) were measured using a spectrofluorometer (FP-8200, JASCO). The absorbance was read at 504 nm and 524 nm. The total ROS content was calculated by standard curve of protein which determined by Bradford reagent. The data was reported as the relative of the total ROS unit per mg of protein. 2.5.8 MDA analysis The lipid peroxidation was measured by MDA assay (Verleysen et al., 2004). The three replications of of SEs were weighed (ca. 100 mg) and added with the reaction mixture containing 700 µl of deionized water and 750 µl of TBA reagent (0.5% (w/v) thiobarbituric acid (TBA) in 20% (w/v) trichloroacetic acid (TCA)). And then, the samples mixture was boiled at 95 °C for 25 min, rapidly cooled on ice (5 min) and then centrifuged at 1000X g (10 min). 30 The absorbance was measured at 532 and 600 nm against TBA reagent (the blank). The concentration of MDA is calculated using the extinction coefficient of MDA (155 mM −1 cm−1) from Beer–Lambert’s equation (Health and Packer, 1968). The MDA contents was compared between the AA-treated and the non AA-treated SEs. 2.6 Statistical analysis The experiment of somatic embryogenesis and cryopreservation were designed using a completely randomized design (CRD). Ten replicates were prepared for somatic embryogenesis and three replicates were used in each treatment of cryopreservation. The mean values were subjected to analysis of variance (ANOVA) and separated using Duncan’s multiple range tests (DMRT) at P ≤ 0.05. A three-way ANOVA was used for a comparison effect between the variables during the interaction of the step in cryopreservation. Statistical analysis was performed using SPSS statistics software. 31 CHAPTER 3 RESULTS 3.1 In vitro cloning via direct somatic embryogenesis 3.1.1 SEs induction SE were formed on solid MVW medium supplemented with NAA only (0.1 and 0.3 -1 mgl ) and 0.1 mgl-1 NAA in combination with TDZ (0.1, 0.5 and 1 mgl-1) after a culture period of 3 months. Among these treatments, the percentage of SE formation of 50-68.33% (Table 9) showed no statistically significant difference. Maximum number of SE per explant (5.19 SEs/explant) was obtained on this basal medium supplemented with 0.1 mgl-1 NAA. However, number of SE per explant tended to decrease after culturing on media containing 0.1 mgl -1 NAA combined with higher concentrations (0.1-0.5 mgl-1) of TDZ. Moreover, the treatment of 0.1 mgl-1 NAA also provided survival rate of 87.5%, which was on par with the control (95%), and were greater than those of other treatments. 32 Table 9 Effects of NAA and TDZ on SE induction of Paphiopedilum niveum. Data derived from four-month-old protocorms cultured on MVW medium supplemented with various concentrations of PGRs and grown in darkness for 3 months. PGR (mgl-1) Survival rate SE formation Number of SEs (%) (%) per explant 0 95.00±3.33a 0.00±0.00b 0.00±0.00c 0 0.1 52.50±7.86bcd 0.00±0.00b 0.00±0.00c 0 0.5 45.00±6.23bcdef 0.00±0.00b 0.00±0.00c 0 1 50.00±7.45bcde 0.00±0.00b 0.00±0.00c 0.1 0 87.50±4.16a 68.33±11.77a 5.19±0.67a 0.1 0.1 55.00±8.98bcd 50.00±15.11a 4.60±0.78ab 0.1 0.5 60.00±5.53bc 61.67±13.62a 3.38±0.78ab 0.1 1 40.00±4.08def 53.33±11.86a 2.67±0.75b 0.3 0 62.50±4.17b 60.00±12.72a 4.52±0.82ab 0.3 0.1 57.50±3.82bcd 0.00±0.00b 0.00±0.00c 0.3 0.5 30.00±6.24f 0.00±0.00b 0.00±0.00c 0.3 1 27.50±4.49f 0.00±0.00b 0.00±0.00c 0.5 0 42.50±3.82cdef 0.00±0.00b 0.00±0.00c 0.5 0.1 30.00±3.33f 0.00±0.00b 0.00±0.00c 0.5 0.5 35.00±4.08ef 0.00±0.00b 0.00±0.00c 0.5 1 55.00±6.24bcd 0.00±0.00b 0.00±0.00c NAA TDZ 0 Values shown above represent the mean±standard error (S.E.). Comparison of the mean values was analyzed using the Duncan’s Multiple Range Test (DMRT). Values with different letters indicate significant differences at P ≤ 0.05. 33 3.1.2 SEs proliferation One-month old protocorm-derived SEs cultured on PGR-free medium in light conditions were grown on solid MVW supplemented with NAA (0, 0.1, 0.3 and 0.5 mgl-1), or in combination with kinetin (0, 1, 5 and 10 mgl-1). The largest increase in fresh weight (FW) of SE (183.33 mg/100 mg of initial fresh weight) and maximum survival rate (88.33%) were obtained on the PGR-free control media (Table 10). The combination of NAA and kinetin appeared to lower the increased of FW in comparison to the control, and some treatments exhibited undesirable effects. For instance, the use of 0.3 mgl-1 NAA combined with 1 or 5 mgl - 1 kinetin showed a decrease in percentage of survival, and the deterioration of color of the SE. Table 10 Effects of NAA and kinetin on SE proliferation, survival, and color of the regenerated SEs of Paphiopedilum niveum. Data derived from SE clumps cultured on MVW supplemented with single and combination of NAA and kinetin after culture for 2 months PGR (mgl-1) Increased FW (mg)/ NAA kinetin 0 0 Survival (%) 100 mg initial FW 183.33±28.93a cd 88.33±0.77a (Color of SEs) Green ab Green 0 1 62.32±23.49 0 5 93.44±29.79abcd 78.33±1.03ab Green 0 10 93.55±34.96abcd 55.00±1.35ab Yellow 0.1 0 158.05±34.76abc 83.33±1.11ab Green ab Green abcd 80.00±1.02 Visual observation 0.1 1 128.85±20.87 0.1 5 82.25±27.94bcd 80.00±0.83ab Green 0.1 10 168.96±36.40ab 86.67±0.99a Green 0.3 0 56.90±20.51d 80.00±1.10ab Green abcd 80.00±0.83 0.3 1 147.18±34.36 0.3 5 93.42±25.80abcd 46.67±1.38b Yellow 0.3 10 122.38±19.43abcd 73.33±1.16ab Green 0.5 0 95.78±17.38abcd 60.00±1.43ab Yellow abcd 50.00±1.18 ab 76.67±1.19 Yellow ab Green 0.5 1 140.48±42.27 0.5 5 127.70±35.97abcd 76.67±1.19ab Green 0.5 10 109.00±23.92abcd 76.67±1.00ab Green Values shown above represent the mean±standard error (S.E.). Comparison of the mean values was analyzed using the Duncan’s Multiple Range Test (DMRT). Values with different letters indicate significant differences at P ≤ 0.05, FW: fresh weight 34 3.1.3 Developmental pattern via direct somatic embryogenesis (DSE) Small protruding shoots of SEs (the primary SEs) were observed on four-month-old protocorms cultured on MVW containing 0.1 mgl-1 NAA in the darkness for 2 months (Fig. 8C). These SEs eventually developed to form the obvious character of SEs, with distinctive shoots showing within 3 months of culture (Fig. 8D). The clusters of primary SEs also exhibited healthy green shoots after being transferred onto PGR-free MVW under light for a month (Fig. 8E). Two months after the transfer, secondary SEs were observed on the primary SEs (Fig. 8F). The primary and secondary SEs with well-developed shoots were then excised into single SE for use as explants, and were subsequently transferred to the plantlet regeneration medium (MVW containing 2 gl-1 activated charcoal and 50 gl-1 banana homogenate). The shoots, followed by autonomous rooting without any PGRs, were obtained 4 months after the subculturing. The plantlets grew well under the greenhouse condition (Fig. 8G). Histological observations revealed that four-month-old protocorms were comprised of two portions of parenchymatous tissues, where the cells of the upper part of protocorm were smaller meristematic cells with large nuclei compared to those at the basal part (Fig. 9A). At the initial stage of culture on SE induction medium, cell division of proembryogenic cells were observed at the surface of the protocorm without an intervening callus phase. This is represented by the densely stained cells with large nuclei (Fig. 9B). The division of subepidermal and epidermal cells at basal layer were limited, and enabled the unequal expansion of cell layers which gave rise to the embryo proper (Fig. 9C). Moreover, the histodifferentiation indicated that the regeneration process was direct somatic embryogenesis (DSE), which had occurred within 1 month of culture, with the embryogenic masses subsequently developing into the ‘globular’ stage in the second month of culture (Fig. 9D). At the third month, the enlargement of multiple primary SE was observed at the basal or lateral part of the original protocorm (Fig. 9E). After these primary SEs were transferred to the media for SE proliferation, , the secondary SE originating on the surface of the primary SE was then observed after culturing on PGR-free MVW for 2 months (Fig. 9F). After that, the ex vitro plantlet presented the well developed of root system under green house condition (Fig. 9G). 35 Figure 8 Morphological characteristics during SE formation and proliferation of Paphiopedilum niveum. (A) Flowering plant. (B) Four-month-old protocorm with a small shoot. (C) Early stages of primary SEs (arrows) developing directly from protocorms cultured on MVW + 0.1 mgl-1 NAA for 2 months in darkness. (D) Cluster of 3 month old SEs with distinctive shoots (arrows) cultured on MVW + 0.1 mgl-1 NAA (E) Original protocorm (P0) and primary SEs (P1) exhibiting healthy green shoots after culture on PGR-free MVW for 1 month under light conditions. (F) Secondary SEs (P2) proliferating from the tissue of primary SEs (P 1) growing on the original protocorm (P0) after a further 2 months of culture on PGR-free MVW. (G) Vigorous regenerated plantlets after removal from flasks and grown under greenhouse conditions for 4 months Figure 9 Histological observation on direct somatic embryogenesis (DSE) of Paphiopedilum niveum. (A) Four-month-old protocorm exhibiting single meristem region at shoot pole (arrow). (B) Protocorm showing the initiation of new meristematic tissue, indicated by active dividing cells with densely stained cytoplasm (inset and arrow) after one month of culture on MVW + 0.1 mgl-1 NAA. (C) Developing SE (globular in shape) represented by the meristematic cells at the epidermal and subepidermal layers (arrows) with heavily stained cytoplasm. (D) Primary SE (arrow) exhibiting unequal division of meristemoids emerging from the protocorm surface after culturing for 2 months on MVW + 0.1 mgl-1 NAA. (E) Primary SEs (P1) (arrows) developing at the lateral zone of basal part of the original protocorm (P 0) 3 months after culturing on MVW + 0.1 mgl-1 NAA. (F) Cluster of secondary 36 SEs (P2) developing on the primary SEs (P1) which were formed on the original protocorm (P0) after a further 2 months of culture on PGR-free MVW. 37 3.1.4 Genetic variation assessment using RAPD Ten selected RAPD primers were used in the genomic DNA amplification. The assessment revealed that there was no variation between the in vitro mother plant (V1) and the regenerant clones (V2 and V3), as indicated by the identical banding patterns in all the samples (Fig. 10). The results showed a number of scored reproducible bands in clones 1, 2 and 3 of 102, 91 and 98 bands, respectively (Table 1). The total numbers of bands in the V1–V3 plantlets of clone 1 (306 bands), clone 2 (273 bands) and clone 3 (294 bands) were analyzed. The sizes of the amplified bands, which the primers chosen could be separated distinctively, were in the range of 150 to 2,500 bp. Figure 10 Gel electrophoresis of RAPD from clone 1–3 of Paphiopedilum niveum showing monomorphic banding patterns generated using ten selected primers. The mother plant is represented by (V1), and the regenerant plants by (V2 and V3) 38 Figure 11 Diagrammatic summary of in vitro cloning of genetically uniform Paphiopedilum niveum via direct somatic embryogenesis (DSE). (A) Seeds were collected from five-monthold capsule. (B) Pretreatment with 1% (v/v) clorox solution for 60 min. (C) Pretreated seeds were cultured in liquid PGR-free MVW in the darkness for 4 months. (D) Four-month-old protocorms were cultured on SE induction medium (MVW containing 0.1 mgl -1 NAA) for 3 months to induce DSE, (E) SE clump proliferated after culture on SE proliferation medium (PGR-free MVW) for 2 month. (F) SE has formed a plantlet after culture on MVW containing 0.2% activated charcoal and 50 mgl-1 banana homogenate for 4 months. (G) Genetic fidelity of in vitro plantlets (V2 and V3) was examined by RAPD analysis in comparison with the mother plant (V1). (H) Four-month-old plantlets have acclimatized and grew well under greenhouse conditions. 39 3.2 Cryopreservation by V cryo-plate method 3.2.1 The optimization of the V cryo-plate method and water content determination According to the highest survival percentage of non-cryopreserved (46.67±23.09%) and cryopreserved SEs (20.00±10.33%), the suitable conditions (treatment no. 18, Table 11) for V cryo-plate method of this orchid species were suggested. The appropiate conditions were as follows: samples were in 0.6 M sucrose (1 d) for the 2nd preculture followed by 1.2 M sucrose in LS for 30 min, and then being transferred to exposure time with PVS2 for 60 min. Based on 3-way ANOVA with no significant interaction between/among these mean values (preculture, osmoprotection and dehydration), PVS2 with exposure time’ (dehydration period) was the crucial factor affecting the survival percentage of SEs (Table 12). Thus, a shorter period of time in PVS2 (dehydration period) for 30 and 45 min showed lower survival percentage in comparison to the suitable treatment (60 min). The WC after 60 min PVS2 dehydration provided the significant decrease to 19.44±2.29% which was lower than that after 30 min (26.94±0.76%) and 45 min (24.71±1.60%) PVS2 dehydration (Fig. 12). 40 Table 11 Factors (2nd preculture, osmoprotection and dehydration) affecting the survival percentage of Paphiopedilum niveum SEs. Data were taken after culture on regrowth medium for 14 days Steps in V cryo-plate Preculture Treatment Sucrose (M) No. Osmoprotection Dehydratio n Sucrose (M) LS containing Survival (%) PVS2 -LN +LN 30 13.33±6.67abc 0.00±0.00b 45 6.67±6.67bc 0.00±0.00b 3 60 0.00±0.00c 0.00±0.00b 4 30 33.33±6.67abc 0.00±0.00b 45 16.67±16.67abc 0.00±0.00b 6 60 6.67±6.67bc 0.00±0.00b 7 30 20.00±0.00abc 0.00±0.00b 45 0.00±0.00c 0.00±0.00b 60 6.67±6.67bc 0.00±0.00b 30 25.39±12.99abc 0.00±0.00b 45 5.57±5.57bc 0.00±0.00b 12 60 35.56±9.87ab 0.00±0.00b 13 30 6.67±6.67bc 0.00±0.00b 45 6.67±6.67bc 0.00±0.00b 15 60 0.00±0.00c 0.00±0.00b 16 30 20.00±11.55abc 0.00±0.00b 45 0.00±0.00c 0.00±0.00b 60 46.67±23.09a 20.00±10.33a 1st* 2nd* 2 M glycerol + sucrose** 1 2 0.4 5 0.4 8 9 10 0.8 1.2 0.2 11 14 17 0.4 0.6 0.8 1.2 18 exposure time (min) The means (mean±S.E.) in column followed by different letters are significantly different at P ≤ 0.05 with DMRT *: preculture for a day, **: osmoprotection for 30 min; -LN: non-cryopreserved SEs; +LN: cryopreserved SEs via V cryo-plate method 41 Table 12 Three-way ANOVA with interaction between all combinations of three main factors (preculture; P, osmoprotection; O and dehydration; D) exhibiting in non cryopreserved Paphiopedilum niveum SEs via V cryo-plate method Factors DF MS F P Preculture (P) 1 600.00 1.898 ns Osmoprotection (O) 2 7.41 0.023 ns Dehydration with PVS2 (D) 2 1258.91 3.983 * Interaction P X O 2 822.22 2.606 ns Interaction P X D 2 921.50 2.916 ns Interaction O X D 4 590.30 1.868 ns Interaction P X O X D 4 201.22 0.637 ns Error 36 Total 53 DF: Degree of freedom; MS: Mean square; F: F-ratio * indicated significant difference at P ≤ 0.05, ns: non-significant difference 42 Figure 12 Percentage of water content in Paphiopedilum niveum SEs during cryopreservation using V cryo-plate method. Data were taken from the optimized protocol (treatment 18). Means with the same letter are not significantly different at P ≤ 0.05 as determined by DMRT. 3.2.2 Effect of ascorbic acid (AA) supplementation on total ROS, MDA level, and survival of AA treated-SEs during the optimized V cryo-plate method In our study, the AA supplementation was performed on day 7 of the culture (before the start of the 1st preculture), the total ROS and MDA levels were determined during the steps of the optimized V cryo-plate protocol, namely; precondition, the 1 st preculture, the 2nd preculture, osmoprotection and dehydration. The non AA-treated SEs was used as a control. The survival percentage and visual observation were taken after 14 d of culture on regrowth medium. The normal level of total ROS (8.09±1.12 ROS unit/ µg protein) was obtained from the intact SE. In the case of non AA-treated SEs, the increase of total ROS level was initially detected at precondition step (11.54±1.02 unit/ µg protein) and reached to the maximum level at the 1st preculture (17.18±0.52 ROS unit/ µg protein) (Fig. 13A). Thereafter, the ROS level, which was high at the 1st preculture, was gradually decreased to normal level at osmoprotection step and then markedly increased again at dehydration step. This result indicated that the arising of oxidative stress occured in the precondition step. In contrast, AA-treated SEs showed the low level of ROS production through 1st preculture to dehydration step. (Fig. 13A). 43 The result of MDA analysis showed that the MDA level was not significantly different in intact SEs (1.13±0.09 µM MDA/ 100 mg fresh weight) and SEs at the precondition step (1.08±0.07 µM MDA/ 100 mg fresh weight). Meanwhile, the continuous increase of MDA level was found from the 1st preculture to dehydration step which showed the highest MDA level in osmoprotection (8.34±0.11 µM MDA/ 100 mg fresh weight) (Fig. 13B). In contrast, the efficiency of AA treatment could efficiently reduce and stabilize MDA level from the 1st preculture to dehydration in AA-treated SEs. The reduction of total ROS and MDA level enabled improvement the survival rate in AA-treated non-cryopreserved SE (42.86±6.15%) and AA-treated cryopreserved SE (39.04±8.50%) which was significantly higher than noncryopreserved (25.71±6.85%) and cryopreserved (8.57±3.40%) of non AA-treated SEs (Fig. 14). From visual observation, non AA-treated cryopreserved SEs exhibited the browning (Fig. 14A) while AA-treated cryopreserved SEs presented white color of the viable shoot after culture on regrowth medium for 14 days (Fig. 14B). The optimal V cryo-plate protocol for P. niveum SEs was illustrated in Figure 15. 44 Figure 13 Determination of (A) total ROS and (B) MDA level during the optimized V cryoplate method of non AA-treated and AA-treated cryopreserved Paphiopedilum niveum SEs. Application of ascorbic acid (AA) at 0.1 mM was done at the 7th day precondition (arrow). The optimized V cryo-plate method comprised of (1) precondition (MVW containing 0.1 M sucrose for 7 d), (2) two steps of preculture (MVW containing 0.2 M (1 st preculture) and 0.6 M (2nd preculture) for 1 d each), (3) osmoprotection (loading solution containing 1.2 M sucrose for 30 min) and (4) dehydration (exposure time to PVS2 for 60 min at 25 °C). Means±S.E. with the same letter are not significantly different at P ≤ 0.05 as determined by DMRT. 45 Figure 14 Survival percentage of non AA-treated and AA-treated Paphiopedilum niveum SEs compared to non-cryopreserved control SEs. Visual observation presenting (A) the browning of non AA-treated cryopreserved SE in comparison with (B) a lutescent shoot of viable AAtreated cryopreserved SE after culture on regrowth medium for 14 days. Means±S.E. with the same letter are not significantly different at P ≤ 0.05 as determined by DMRT. AA: ascorbic acid (0.1 mM) application, Non LN: Non cryopreservation, LN: cryopreservation in liquid nitrogen. 46 Figure 15 Schematic diagram of cryopreservation of Paphiopedilum niveum SEs using V cryoplate method. (A) SEs were preconditioned on PGR-free MVW containing 0.1 M sucrose (7 d), followed by (B) the 1st preculture in MVW containing 0.2 M sucrose (1 d) and (C) the 2 nd preculture in MVW containing 0.6 M sucrose for 1 d. (D) Precultured SEs were embedded onto cryo-plate using 2% alginate gel. (E) Cryo-plates were immersed into LS containing 1.2 M sucrose for 30 min, (F) dehydrated by PVS2 for 60 min (G) Cryotube containing cryo-plate was affixed on cryocane and then (H) plunged into LN. (I) Cryopreserved SEs were thawed in 1 M sucrose solution and (K) cultured on regrowth medium (Fe-free MVW medium containing 0.1 mgl-1 NAA, 0.2% (w/v) PVP-40 and 0.2% (w/v) AC) for 7 d in the darkness and then transferred to light condition. 47 CHAPTER 4 DISSCUSSION 4.1 In vitro cloning by direct somatic embryogenesis 4.1.1 SEs induction The application of NAA at lower concentration (<0.1 mgl -1), either singly or in combination with BA, has reportedly been able to induce organogenesis in many plant species. For instance, direct organogenesis has been reported after the leaf explant of Hydrangea macrophylla was cultured on B5 medium containing 0.05 mgl-1 NAA with 2.25 mgl-1 BA for 50 days (Liu et al., 2011). Gupta et al., (2013) also reported that the direct rhizogenesis (derived from leaf explant) and multiple shoot regeneration (originated from stolon) of Glycyrrhiza glabra could be induced on liquid and solid MS supplemented with 0.01 mgl-1 NAA only. In orchid species, direct somatic embryogenesis of Oncidium flexuosum (15-80%) could be induced from leaf explant after culture on ½ MS containing lower concentration (0.25 µM or 0.05 mgl-1) of NAA in combination with 1.5-13.5 µM (0.33-2.97 mgl-1) TDZ under the darkness (Mayer et al., 2010). Parthibhan et al., (2018) also reported that tTCL stem of Dendrobium aqueum culturing on ½ MS containing very low (0.01 mgl-1) NAA combined with 0.5 mgl-1 BA could stimulate both indirect somatic embryogenesis (11.67%) and direct somatic embryogenesis (10%). Ahamed Sherif et al., (2018), however, revealed that the single use of 0.5 mgl-1 NAA promoted the potential for induction of nodal-derived direct somatic embryogenesis (36.5%) of Anoectochilus elatus. No evidence on SE induction of Paphiopedilum niveum using NAA at lower concentration has been reported prior to our current study, where we now report that the supplementation of NAA at low concentration (0.1 mgl-1) could promote the SE formation in this species. Various types of orchid explants have previously been reported to form SEs in response to the exogenous auxin and cytokinin. Chen and Chang (2001) reported that the use of ½ MS supplemented with 0.3 and 1 mgl-1 IAA, or only 0.3 mgl-1 NAA, was sufficient to induce the direct somatic embryogenesis (DSE) from the leaf of Oncidium ‘Gower Ramsey’, while a single cytokinin application (TDZ, BA, zeatin, and BA) showed greater results in terms of percentage of somatic embryogenesis formation, and number of SEs per explant. In addition, Spathoglottis plicata SEs could be induced from the rhizome-like structures after culture on Phytamax™ orchid maintenance medium containing 2 µM 2-4,D or 0.75 µM IAA for 35 days (Novak and Whitehouse, 2013). Some orchids required the combination of auxin and cytokinin to promote DSE induction. For instance, the DSE of Cymbidium bicolor (Mahendran and Bai, 2012) and 48 Anoectochilus elatus (Ahamed Sherif et al., 2018) could be induced from nodal segments cultured on modified Mitra medium supplemented with 4.54 μM TDZ and 2.69 μM NAA, and also from protocorms cultured on ½ MS containing 1.0 mgl-1 BAP and 2.0 mgl-1 2-4,D. Our results show that low concentrations of NAA (0.1 mgl-1) had a potential to induce DSE in P. niveum, while the combination of NAA and TDZ reduced the number of SEs as TDZ concentration increases (Table 9). The latter is consistent with the results of Vogel and Macedo (2011), who reported that the number of Cyrtopodium glutiniferum SEs decreased significantly after culturing on media containing high TDZ (5 µM) when compared with low TDZ (1 µM) treatments. Malabadi et al., (2004) also demonstrated similar results that higher concentrations of TDZ (13.62-22.71 µM) in VW could decrease SEs numbers of Vanda coerulea compared to those treated with 11.35 µM TDZ. Guo et al., (2011) revealed that the possibility of TDZ being an inhibitor of cytokinin oxidase leads to stimulate the accumulation of endogenous purinebased cytokinin (BA). This event may be responsible for the suppression of embryogenesis in some species such as orchard grass (Dactylis glomerata) and wheat (Triticum aestivum) (Van Staden et al., 2008), and in Cassava (Manihot esculenta) (Wongtiem et al., 2011). However, Zeng et al., (2013) revealed that seed-derived protocorms of Paphiopedilum hangianum could be induced to form the DSE after culture on modified ½ MS supplemented with 5 mgl-1 kinetin and 2 mgl-1 BA for 90 days. These reports demonstrated that the different types of explant sources, as well as the type and concentration of PGRs used, were essential factors for SE formation that involves the dedifferentiation of the somatic cells (Ji et al., 2 0 1 1 ) . Our results has now demonstrated that DSE induction from young protocorm of P. niveum can also be triggered even with low auxin concentrations. 4.1.2 SEs proliferation According to our study, the maximum increased FW was found in the control (freePRGs medium) but the combination of NAA and kinetin did not presented the suitable result. The combination of NAA and kinetin provided positive results in SE proliferation of Dendrobium officinale when cultured on MS medium supplemented with 1 mgl-1 kinetin, 0.2 mgl-1 NAA and 10% coconut water (Zhang et al., 2011). However, our results demonstrated the single application of high NAA (0.5 mgl-1) and high kinetin (10 mgl-1) reduced the survival rate of the SE of P. niveum, and resulted in a change of SE color from green to yellow compared to those cultured on lower concentrations of both NAA and kinetin. Our results were also consistent with Chen et al., (2002), who reported that the use of high kinetin concentrations (4.65 µM) could decrease the number of SEs in Epidendrum radicans as compared with lower 49 kinetin concentrations (1.39 µM). In addition, Ng and Saleh (2011) reported that the SE proliferation pattern of P. rothschildianum exhibited autonomous proliferation of secondary SEs from primary SEs, which occurred after a transfer from ½ MS medium containing low kinetin (0.86 mgl-1) onto PGR-free ½ MS medium supplemented with 60 gl-1 banana homogenate. Our results showed that SEs could be proliferated from young protocorms cultured on MVW containing 0.1 mgl-1 NAA and subsequently transferred to PGR-free MVW. The increase of endogenous auxin accumulation via exogenous auxin supplementation has been reported to be necessary for the early stage of SE formation (Su and Zhang, 2009). Decreasing of auxin level could then allow further proliferation and differentiation of the SE (Yang and Zhang, 2010), and stimulate shoot formation (Novak et al., 2014). 4.1.3 Histological observation The recent study exhibited that proembryogenic cells originated from epidermal and subepidermal layers of P. niveum protocorm without an intervening callus. This histological evidence presented here concerning the newly regenerated SE also coincides with the findings on the investigation of Phalaenopsis orchid somatic embryogenesis by Lee et al., (2013).These regenerated cells then formed a globular-shaped mass and differentiated to the SE, which was similar to SE observed in Cymbidium bicolor (Mahendran and Bai 2012), Tolumnia Louise Elmore ‘Elsa’ (Shen et al., 2018), Phalaenopsis amabilis and Phalaenopsis ‘Nebula’ (Gow et al., 2010). 4.1.4 RAPD analysis RAPD-PCR analysis have been successfully applied to determine the genetic fidelity assessment in various clonal micropropagated plant species, for instance, in three-month-old regenerated plantlets of Dendrobium Second Love cultured ex vitro (Ferreira et al., 2006), and six-month-old in vitro Alhagi maurorum (Agarwal et al., 2015). In our study, RAPD analysis exhibited identical banding patterns, confirming that genetic homogeneity between the mother/original plant (V1) and four-month-old in vitro regenerated plantlets (V2-V3) of P. niveum has been conserved. Krishna et al., (2016) also revaled that in vitro plant regeneration through embryogenesis with lower DNA methylation levels provided better chance of obtaining plants with genetic uniformity than organogenesis pathway. Other important factors that contributed to an increase of somaclonal variations include the type of PGRs selected (Bairu et al., 2011), as well as prolonged culture periods (Martin et al., 2004; Krishna et al., 2016). In the present study, NAA application showed no genetic alteration as compared to strong synthetic PGRs (TDZ and 2,4-D) as revealed by Bairu et al., (2011), as the absorbed NAA can be often 50 converted to the inactivated form, mainly glucosyl ester by enzymatic conjugation with glucose (Machakova et al., 2008; Sauer et al., 2013). However, increased somaclonal variation and epigenetic change have been observed when callus of cocoa (Theobroma cacao) was cultured for an extended period of time (Rodríguez López et al., 2010). 4.2 Cryopreservation by V cryo-plate method In our study, the crucial factor affecting the survival of P. niveum SEs was the exposure time to PVS2 for 60 min (dehydration step) by V cryo-plate method. This condition also revealed that the suitable WC (~20%) was obtained from this PVS2 dehydration for 60 min while short dehydration time (30 and 45 min) gave the unfitting WC (>20%). It was consistent with Engelmann-Sylvestre and Engelmann (2015) who reported that the PVS2 dehydration for 60 min provided 71% survival of cryopreserved Clinopodium odorum shoot tip via V cryoplate method and low survival percentage (>40%) was found after shorter PVS2 dehydration period (10-50 min). It was possible that insufficient dehydration time provided high intracellular WC leading to ice crystallization during cryopreservation at ultra-low temperature (Quain et al., 2009). The high residual WC (>25%), making cryopreserved P. niveum SEs difficult to survive, should be considered for cryopreservation (Chaireok et al., 2016). Sakai (2000) also mentioned that determination of WC was required for minimizing the cryoinjury by the balancing of intracellular WC. . Our results demonstrated that the potential of the single treatment of low (0.1 mM) AA concentration in the critical step (before the arising of oxidative stress) could improve the survival of cryopreserved SEs of P. niveum. No research in determining the total ROS level on survival of any cryopreserved plant material has been reported. Uchendu et al. (2010), however, revealed that MDA level was decreased after 0.28 mM AA supplemented in almost step, namely; preculture, LS and thawing, during cryopreservation of blackberry shoot tip (Rubus hybrida cv. Chehalem and Hull Thornless). In addition, this AA application (multiple treatment) could enhance the survival percentage of these cryopreserved blackberry shoot tips. Meanwhile, the AA treatment without oxidative stress determination by applying 0.28 mM AA in LS step could enhance a few survival percentage (3.3%) of pulasan shoot tip (Nephelium rumboutan-ake) (Chua and Normah, 2011). Ibrahim and Normah (2013) also reported that there were no significant difference between non AA-treated and AA-treated (0.28 mM) cryopreserved mangosteen (Garcinia mangostana) shoot tip at different steps (preculture, LS, PVS2, unloading and recovery). Therefore, this is the first study to indicate that total ROS and 51 MDA level could be considered as indicators required for selection of the appropriate step of AA application during cryopreservation protocol. 52 CHAPTER 5 CONCLUSION This study presented the simple protocol of somatic embryo (SE) induction and proliferation without genetic alteration. The primary SEs could be induced from four-monthold protocorm after culture on solid MVW containing 0.1 mgl-1 NAA for three months (with monthly subculture interval) in the darkness. The culture condition provided 68% of SE formation with the maximum number of SEs around 5 SEs per explant. These primary SEs presented the highest increase in fresh weight of proliferated SEs (183 mg/100 mg initial FW) when cultured on solid PGRs-free MVW for two months. These obtained SEs exhibiting the vigorous character developed to plantlet after being transferred to MVW supplemented with 0.2% AC and 50 mgl-1 BH for four months. The result of RAPD analysis proved that the plantlets which were developed from mother explant (V1) and regenerated plantlets (V2 and V3) showed the uniform genetic. The research also presented the first successful cryopreservation and the improvement in survival of cryopreserved of P. niveum SEs using V cryo-plate method. The optimized V cryo-plate method consisted of 1) SEs (1-1.5 mm Ø) were preconditioned in solid MVW containing 0.1 M sucrose for seven days, 2) and then SEs were treated with the two-step preculture in 0.2 M and 0.6 M sucrose, each with one day, 3) precultured SEs were incubated in LS supplemented with 1.2 M sucrose for 30 min, followed by 4) immersed in PVS2 for 60 min at room tempereture (25 °C). This optimized condition provided ~20% of survival cryopreserved P. niveum SEs. This protocol was improved to increase the survival of cryopreserved SEs by applied 0.1 mM AA on the 7th day of precondition step (before the extreme arising of total ROS and MDA). 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Zhong Yao Cai. 34(8): 1172-1177. 65 APPENDICES 66 APPENDIX A Culture medium Modified Vacin and Went (1949) Micronutrient Macronutrient Stock solution Stock 1 Calcium* Stock 2 FeEDTA Components (NH4)2SO4 mgl-1 500 KH2PO4 250 KNO3 525 MgSO4·7H2O 250 Ca3(PO4)2 200 MnSO4·5H2O 22.3 ZnSO4·7H20 8.6 H3BO3 6.2 KI Na2MoO4·2H2O 0.83 0.25 CuSO4·5H2O 0.25 CoCl2·6H2O 0.25 Na2EDTA 37.25 FeSO4·7H2O 27.85 Glycine Thiamine HCl (B1) Nicotinic acid Vitamins Stock 3 Pyridoxine HCl (B6) Myo-inositol Bacto-peptone sucrose phytagel/gelrite Additives Others activated charcoal 1000 mgl-1 chitosan (MW=80) The pH of medium adjusts to 5.3-5.4 *Dissolved in 1 N HCl 2 0.1 0.5 0.1 100 2,000 20,000 3,000 2,000 5 mll-1 67 APPENDIX B Histological studies Formalin-aceto-alocohol II (FAA II) Components 70% EtOH Glacial acetic acid Formalin Fixation period is 48 h 90 ml 5 ml 5 ml Ethanol-tert-butyl alcohol series (EtOH/TBA series) Component (ml) Total No. 95% 100% alocohol (%) TBA DW EtOH EtOH 1 5 5 95 2 10 10 90 3 20 20 80 4 30 30 70 5 50 10 40 50 6 70 20 50 30 7 85 35 50 15 8 95 55 40 5 9 100 75 25 10 100 11 100 12 - 50 - - - Other eosin paraffin oil (50 ml) DW: distilled water, TBA: tert-butyl alcohol Delafield hematoxylin Stock solution Components Hematoxylin 4g Solution A* 95% EtOH 125 ml Solution B Aluminium alam (NH4)Al(SO4)2 8g Distilled water 400 ml Potassium KMnO4 0.2 g permanganate solution Distilled water 5 ml Delafield Hematoxylin Solution A 200 ml staining solution Solution B 200 ml Glycerine 200 ml Total volume 600 ml Add KMnO4 solution 5 ml in 600 ml of staining solution and incubate for 2 days *dissolve in hot plate and filter 68 Safranin Components Safranin O Methyl cellosolve (ethylene glycol monoethyl ether) 95% EtOH Sodium acetate formalin Total volume 2g 100 ml 50 ml 2g 4 ml 145 ml Deparafifinization and rehydrateion process (Ruzin, 1999) Immerse slide with paraffinized section in the following protocol 1. Xylene substitute I 10 min 2. Xylene substitute II 10 min 3. Absolute ethanol : Xylene substitute 5 min 4. Absolute ethanol I 2 min 5. Absolute ethanol II 2 min 6. 95% ethanol I 2 min 7. 95% ethanol II 2 min 8. 70% ethanol I 2 min 9. 70% ethanol II 2 min 10. Distilled water Delafield’s hematoxylin and safranin O staining (Ruzin, 1999) 1.Stain in Delafield’s hematoxylin 20-30 min 2. Rinse in distilled water 2 min 3. Acidulated water 10 sec (1-2 drops of HCl / 100 ml distilled water) 4. Rinse in distilled water 10 dips 5. Immerse into 0.1% (w/v) Li2CO3 2 min 6. Counterstain with safranin O >3 min 7. Rinse in acidulated water 2 dips 8. Place into 0.1% (w/v) Li2CO3 2 min 9. Rinse with tap water 2 dips 10. Pass through 50% EtOH I, II A few minutes in each strength 12. Pass through 70% EtOH I, II A few minutes in each strength 13. Pass through 95% EtOH I, II A few minutes in each strength 14. Pass through absolute EtOH I, II A few minutes in each strength 15. Pass through absolute EtOH : Xylene substitute A few minutes in each strength I, II (1:1) 16. Pass through Xylene I, II A few minutes in each strength 17. Mount Result: Nucleus = Blue; Cytoplasm = Pink 69 APPENDIX C RAPD analysis dNTP Components Each of 100 mM deoxynucleotide (dATP, dGTP, dCTP and dTTP) Deionized water Mix 100 µl of each deoxynucleotide to prepare 5 mM dNTP 50 µl 950 µl Primer (10 µM) Components Each of 100 mM primer Deionized water 5 µl 45µl Mixture for PCR reaction Components 20 ng of template DNA dNTP Primer ThermoPolTM buffer Taq DNA polymerase deionized water Total volume 1 µl 2 µl 1.5 µl 2.5 µl 0.25 µl 17.5 µl 24.5 µl The PCR reaction total cycling at 40 cycles 70 APPENDIX D V cryo-plate method 2% (w/v) Na-alginate solution Components Stock 1 of MVW Ca3(PO4)2 2% (w/v) Na-alginate 0.4 M sucrose Distilled water The pH of solution adjusts to 5.3-5.4 CaCl2 solution Components Stock 1 of MVW Ca3(PO4)2 0.1 M CaCl2 0.4 M sucrose Distilled water The pH of solution adjusts to 5.3-5.4 10 ml 20 mg 2g 13.662 g Adjust to the final volume to 100 ml 100 ml 200 mg 11.098 g 136.62 g Adjust to the final volume to 1000 ml Loading solution (LS) Sucrose concentration in LS 0.4 M LS 0.8 M LS 1.2 M LS Stock 1 of MVW 100 ml 100 ml 100 ml Ca3(PO4)2 200 mg 200 mg 200 mg Sucrose 136.92 g 273.84 g 410.76 g 2.0 M glycerol 184.18 g 184.18 g 184.18 g Distilled water Adjust to the final volume to 1000 ml The pH of solution adjusts to 5.3-5.4 Components Plant Vitrification Solution 2 (PVS2) Components Stock 1 of MVW Ca3(PO4)2 0.4 M Sucrose 15% (w/v) ethylene glycol 15% (w/v) DMSO 30% (w/v) glycerol Distilled water The pH of solution adjusts to 5.3-5.4 DMSO: Dimethyl sulfoxide 100 ml 200 mg 136.62 g 150 g 150 g 300 g Adjust to the final volume to 1000 ml 71 Unloading solution (1 M sucrose solution) Components Stock 1 of MVW 100 ml Ca3(PO4)2 200 mg 1.0 M sucrose 342.30 g Distilled water Adjust to the final volume to 1000 ml The pH of solution adjusts to 5.3-5.4 Rinsing solution Components Stock 1 of MVW Ca3(PO4)2 Stock 2 of MVW Stock 3 of MVW Sucrose PVP-40 Distilled water The pH of solution adjusts to 5.3-5.4 PVP-40: Polyvinylpyrrolidone-40 Precondition medium Components Stock 1 of MVW Ca3(PO4)2 Stock 2 of MVW Stock 3 of MVW FeEDTA 0.1 M Sucrose phytagel/gelrite 1000 mgl-1 chitosan (MW=80) Distilled water The pH of solution adjusts to 5.3-5.4 100 ml 200 mg 0.5 ml 1 ml 20 g 2g Adjust to the final volume to 1000 ml 100 ml 200 mg 0.5 ml 1 ml 10 ml 34.23 g 3g 5 ml Adjust to the final volume to 1000 ml 72 Preculture medium Components Stock 1 of MVW Ca3(PO4)2 Stock 2 of MVW Stock 3 of MVW FeEDTA Sucrose 1000 mgl-1 chitosan (MW=80) phytagel/gelrite Distilled water The pH of solution adjusts to 5.3-5.4 Regrowth medium (Fe-free) Components Stock 1 of MVW Ca3(PO4)2 Stock 2 of MVW Stock 3 of MVW 2 % Sucrose PVP-40 activated charcoal 1000 mgl-1 chitosan (MW=80) phytagel/gelrite Distilled water The pH of solution adjusts to 5.3-5.4 PVP-40: Polyvinylpyrrolidone-40 Sucrose concentration 0.4 M sucrose 0.6 M sucrose 100 ml 100 ml 200 mg 200 mg 0.5 ml 0.5 ml 1 ml 1 ml 10 ml 10 ml 136.92 g 205.38 g 5 ml 5 ml 3g 3g Adjust to the final volume to 1000 ml 100 ml 200 mg 0.5 ml 1 ml 20 g 2g 2g 5 ml 3g Adjust to the final volume to 1000 ml 73 APPENDIX E Oxidative stress determination ROS determination 10 mM Tris-buffer List of chemical Tris-HCl Deionized water The pH of solution adjusts to 7.2 15.76 g 100 ml 10 mM 2',7' –dichlorofluorescin diacetate (DCFDA) List of chemical DCFDA 4.8527 mg DMSO 1 ml DMSO: Dimethyl sulfoxide Dilute to 1 mM DCFDA for working solution ROS determination method The fluorescence value of sample against control is compare with a total protein concentration of the sample which determined by Bradford reagent. 74 1 mgml-1 BSA List of chemical BSA 5 mg Deionized water 5 ml BSA: Bovine serum albumin Construction method of standard curve of protein concentration Serial dilution of BSA BSA: Bovine serum albumin, DI: Deionized water 75 MDA determination TBA reagent (Verleysen et al., 2004) List of chemical Thiobarbituric acid (TBA) Trichloric acid (TCA) Deionized water 0.5 g 20 g 100 ml MDA analysis Method Beer Lambert’s equation (Heath and Packer, 1968) 𝑨 C= 𝑳𝜺 A = absorbance C = Concentration (moll-1) L = path length ɛ = extinction coefficient (mM-1cm-1) *extinction coefficient of MDA = 155 mM-1cm-1 76 APPENDIX F Data from statistical analysis SEs induction Survival percentage Duncana Subset for alpha = 0.05 VAR000 01 N 1 2 3 4 5 6 13.00 10 27.5000 12.00 10 30.0000 14.00 10 30.0000 15.00 10 35.0000 35.0000 10.00 10 40.0000 40.0000 40.0000 4.00 10 42.5000 42.5000 42.5000 42.5000 6.00 10 45.0000 45.0000 45.0000 45.0000 45.0000 7.00 10 50.0000 50.0000 50.0000 50.0000 5.00 10 52.5000 52.5000 52.5000 8.00 10 55.0000 55.0000 55.0000 16.00 10 55.0000 55.0000 55.0000 11.00 10 57.5000 57.5000 57.5000 9.00 10 60.0000 60.0000 3.00 10 2.00 10 87.5000 1.00 10 95.0000 Sig. 62.5000 .053 .088 .056 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 10.000. .056 .056 .337 77 SE formation (%) Duncana Subset for alpha = 0.05 VAR000 01 N 1 2 1.00 10 .0000 4.00 10 .0000 5.00 10 .0000 6.00 10 .0000 7.00 10 .0000 11.00 10 .0000 12.00 10 .0000 13.00 10 .0000 14.00 10 .0000 15.00 10 .0000 16.00 10 .0000 8.00 10 50.0000 10.00 10 53.3330 3.00 10 60.0000 9.00 10 61.6660 2.00 10 68.3330 Sig. 1.000 .117 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 10.000. 78 Number of SEs per explant Duncana Subset for alpha = 0.05 VAR00 001 N 1 2 3 1.00 10 6.00 10 2.6667 5.00 10 3.3750 3.3750 3.00 10 4.5238 4.5238 4.00 10 4.6000 4.6000 2.00 10 Sig. .0000 5.1905 1.000 .060 .078 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 10.000. 79 SEs proliferation Increased fresh weight (mg)/ 100 mg initial fresh weight Duncana Subset for alpha = 0.05 VAR0000 1 N 1 2 3 4 9.000 10 56.89600 2.000 10 62.31700 62.31700 7.000 10 82.25200 82.25200 82.25200 11.000 10 93.42000 93.42000 93.42000 93.42000 3.000 10 93.43500 93.43500 93.43500 93.43500 4.000 10 93.54900 93.54900 93.54900 93.54900 13.000 10 95.77900 95.77900 95.77900 95.77900 16.000 10 109.00000 109.00000 109.00000 109.00000 12.000 10 122.37900 122.37900 122.37900 122.37900 15.000 10 127.69700 127.69700 127.69700 127.69700 6.000 10 128.85200 128.85200 128.85200 128.85200 14.000 10 140.48400 140.48400 140.48400 140.48400 10.000 10 147.18300 147.18300 147.18300 147.18300 5.000 10 158.05300 158.05300 158.05300 8.000 10 168.96400 168.96400 1.000 10 Sig. 183.33300 .076 .059 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 10.000. .089 .078 80 Survival percentage Duncana Subset for alpha = 0.05 VAR0000 1 N 1 2 11.000 10 46.66667 10.000 10 50.00000 50.00000 4.000 10 55.00000 55.00000 13.000 10 60.00000 60.00000 12.000 10 73.33333 73.33333 14.000 10 76.66667 76.66667 15.000 10 76.66667 76.66667 16.000 10 76.66667 76.66667 3.000 10 78.33333 78.33333 2.000 10 80.00000 80.00000 6.000 10 80.00000 80.00000 7.000 10 80.00000 80.00000 9.000 10 80.00000 80.00000 5.000 10 83.33333 83.33333 8.000 10 86.66667 1.000 10 88.33333 Sig. .065 .054 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 10.000. 81 Cryopreservation via V cryo-plate Survival rate of -LN SEs Duncana Subset for alpha = 0.05 VAR0000 1 N 1 2 3 3.000 3 .00000 8.000 3 .00000 15.000 3 .00000 17.000 3 .00000 11.000 3 5.56667 5.56667 2.000 3 6.66667 6.66667 6.000 3 6.66667 6.66667 9.000 3 6.66667 6.66667 14.000 3 6.66667 6.66667 1.000 3 13.33333 13.33333 13.33333 5.000 3 16.66667 16.66667 16.66667 7.000 3 20.00000 20.00000 20.00000 16.000 3 20.00000 20.00000 20.00000 13.000 3 23.33333 23.33333 23.33333 10.000 3 25.39683 25.39683 25.39683 4.000 3 33.33333 33.33333 33.33333 12.000 3 35.55556 35.55556 18.000 3 Sig. 46.66667 .064 .092 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 3.000. .056 82 Survival rate of +LN SEs Duncana Subset for alpha = 0.05 VAR000 01 N 1 2 1.00 3 .0000 4.00 3 .0000 5.00 3 .0000 6.00 3 .0000 7.00 3 .0000 11.00 3 .0000 12.00 3 .0000 13.00 3 .0000 14.00 3 .0000 15.00 3 .0000 16.00 3 .0000 8.00 3 .0000 10.00 3 .0000 3.00 3 .0000 9.00 3 .0000 2.00 3 .0000 17.00 3 .0000 18.00 3 Sig. 20.0000 1.000 .117 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 3.000. 83 Three-way ANOVA with interaction between all combinations of three main factors (preculture; P, osmoprotection; O and dehydration; D) Factors SS DF MS F P Preculture (P) 600.00 1 600.00 1.898 0.000000 Osmoprotection (O) 14.81 2 7.41 0.023 0.176763 Dehydration with PVS2 (D) 2517.81 2 1258.91 3.983 0.976850 Interaction P X O 1644.44 2 822.22 2.606 0.027370 Interaction P X D 1843.00 2 921.50 2.916 0.088048 Interaction O X D 2361.19 4 590.30 1.868 0.067055 Interaction P X O X D 804.89 4 201.22 0.637 0.137397 11378.00 36 316.06 Error Total 53 Water content Duncana Subset for alpha = 0.05 VAR000 01 N 1 2 3 4 5 6 8.000 4 7.000 4 24.70703 6.000 4 26.93827 5.000 4 4.000 4 3.000 4 2.000 4 79.48554 1.000 4 79.63203 Sig. 19.44362 40.31189 54.08435 64.48554 1.000 .322 1.000 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 4.000. 1.000 1.000 .948 84 ROS content of AA-treated and non AA-treated SEs Duncana Subset for alpha = 0.05 VAR0000 1 N 1 2 3 4 8.000 3 5.11041 9.000 3 5.29697 7.000 3 6.33470 10.000 3 6.42727 5.000 3 7.05513 1.000 3 8.08767 2.000 3 6.000 3 12.50886 4.000 3 13.70408 3.000 3 Sig. 8.08767 11.54429 11.54429 13.70408 17.18397 .157 .069 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 3.000. .270 .068 85 ROS content of AA-treated and non AA-treated SEs Duncana Subset for alpha = 0.05 VAR0000 1 N 1 2 3 4 5 2.000 4 1.08279 1.000 4 1.13457 7.000 4 2.19885 2.19885 10.000 4 2.23213 2.23213 9.000 4 8.000 4 3.69621 3.000 4 4.09692 6.000 4 5.98819 4.000 4 6.23047 5.000 4 3.46838 3.46838 8.34320 Sig. .094 .057 .339 .694 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 4.000. VAR00002 Duncana Subset for alpha = 0.05 VAR00001 2 N 1 2 7 8.57143 1 7 25.71429 4 7 39.04762 3 7 42.85714 Sig. .074 Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 7.000. 25.71429 .089 1.000 86 VITAE Name Mr. Sutthinut Soonthornkalump Student ID 5610230044 Educational Attainment Degree Name of Institution Year of Graduation Master of Science Mahidol University 2012 Kasetsart University 2007 (Plant Science) International program Bachelor of Science (Fisheries) Scholarship and Awards during Enrollment - Graduate Studies Scholarship (95000201) of Prince of Songkla University Graduate school, Prince of Songkla University, Thailand - Scholarship for Support Exchange Students and International Credit Transferred through ASEAN Community, Graduate School, Prince of Songkla University, Thailand - International Society of Horticultural Science (ISHS) Student award for the best poster presentation from The First Symposium on Tropical and Subtropical Ornamentals (TSO2016) held in Krabi, Thailand - STEM workforce project National Science and Technology Development Agency (NSTDA), Thailand List of Publication and Proceedings - Soonthornkalump, S. 2016. In vitro conservation: confronting in the age of climate change. Chronica Horticuturae. 56(4): 17-23. - Soonthornkalump, S., Nakkanong, K. and Meesawat, U. In vitro cloning via direct somatic embryogenesis and genetic stability assessment of Paphiopedilum niveum (Rchb.f.) Stein, the endangered Venus’s slipper orchid (Submitted to In Vitro Cellular & Developmental Biology – Plant) - Soonthornkalump, S., Yamamoto, S. and Meesawat, U. The optimization of V cryo-plate protocol and survival improvement for cryopreserved somatic embryos of Paphiopedilum niveum (Rchb.f.) Stein: Influence of ascorbic acid (Submitted to CryoLetters)