Conservation and propagation of an endangered medicinal orchid (anoectochilus setaceus lindl ) by in vitro technique

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY BUI THU UYEN Conservation and propagation of an endangered medicinal orchid (Anoectochilus setaceus Lindl.) by in vitro technique BACHELOR THESIS Study Mode: Full-time Major Faculty Batch : Environmental Science and Management : Advanced Education Program : 2017 – 2021 Thai Nguyen, 06/2021 Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Environmental Science and Management Student name Bui Thu Uyen Student ID DTN 1854290001 Conservation and propagation of an endangered Thesis title medicinal orchid (Anoectochilus setaceus Lindl.) by in vitro technique Supervisor Associate Prof Nguyen Tien Dung Dr.rer.nat Nikki Heherson A Dagamac Supervisor’s signature Abstract: Anoectochilus setaceus Lindl is overexploited and over collected for its value in horticultural, medicinal, and food usage that it is concerned as endangered forest plants (EN) In this study, the most suitable in vitro medium formula for A setaceus Lindl regarding basic medium, BAP and Kinetin concentration and organic compounds examination The medium formula was examined based on the monitoring of shoot regeneration rate and length The most suitable basic medium for conservation and propagation A.setaceus Lindl in vitro is ½ MS Between studied growth regulators, BAP is highly recommended in use, rather than Kinetin, account for its excellent performance with both criteria, especially at 0.8 mg/L BAP, and to avoid the dependence of Kinetin in timing Association of 100 ml/L Coconut water + 10g/L Potato + 10g/L Carrot showed the best performance as organic compound the techniques of in vitro must come along to support in situ conservation of A.setaceus Lindl In order to rehabitat the orchid back into the wild, the collaborations between research institute and conservationist are highly recommended to acquire A stricter conservation governance manages the biodiversity and secures the effectiveness of conservation from being disturbed by human activities, starting with updating the profile of A.setaceus Lindl conservation priority to date ii Keywords Anoectochilus setaceus Lindl, in vitro propagation, conservation Number of pages Date of submission June, 2021 iii ACKNOWLEDGEMENT I would like to extend my gratitude and sincere thanks to my supervisor Associate Prof Nguyen Tien Dung, Faculty of Biotechnology and Food technology, Thai Nguyen University of Agriculture and Forestry Thanks to his dedicate supervision and support, my study has obtained a success I would like to express my gratitude to my co-supervisor Dr.rer.nat Nikki Heherson A Dagamac, Institute of Botany and Landscape Ecology, University of Greifswald He does not only teach me to be a better students and researcher, but also shows me how to be a better person I express my heartfelt thanks to my mom, a wonderful woman who motivates and orients me She is the most supportive person who never lets me walk alone in any journey and who has raised me to be a good person I am honor to be her daughter Along with my study, I have been gratefully inspired by my sister who is such a role model of an empowered, independent woman to me Last, but not the least, I would like to show a great appreciation to my fiancé for his love and support I am blessed to find my best friend, my soulmate and my partner in one person TABLE OF CONTENTS ACKNOWLEDGEMENT iv TABLE OF CONTENTS v LIST OF FIGURES vii LIST OF TABLES viii PART I: INTRODUCTION 1.1 Research rationale 1.2 Research’s objectives 1.3 Significance of the study 1.4 Scope and Limitations PART II LITERATURE REVIEW 2.1 Orchid conservation status 2.1.1 Anoectochilus setaceus Lindl Conservation status 2.2 In vitro 2.2.1 Definition PART III METHODOLOGY 12 3.1 Effects of basal culture medium on shoot regeneration 13 3.1.1 Disinfection of sample 13 3.1.2 Medium preparation 13 3.2 Effects of 6-Benzylaminopurine (BAP) and Kinetin on shoot regeneration 14 3.2.1 Effect of BAP on shoot regeneration 14 3.2.2 Effects of Kinetin in shoot regeneration 14 v 3.3 Effects of some organic compounds (coconut water, potatoes, carrots) on multiplication of cultured samples 15 PART IV RESULTS 16 4.1 Effects of culture medium formula on shoot regeneration 16 4.1.1 Effects of culture medium formula on shoot regeneration rate 16 4.1.2 Effects of culture medium on average regenerated shoot length 17 4.2 Effects of 6-Benzylaminopurine (BAP) and Kinetin in shoot regeneration stage 19 4.2.1 Effects of BAP on shoot regeneration stage 19 4.2.2 Effects of Kinetin on shoot regeneration stage 21 4.3 Effects of some organic compounds (coconut water, potatoes, mashed carrot) on shoot multiplication stage 25 4.3.1 Effects of some organic compounds (coconut water, potatoes, mashed carrot) on shoot multiplication rate 25 4.3.2 Effects of some organic compounds (coconut water, potatoes, mashed carrot) on multiplied shoot length 27 PART V: DISCUSSION AND CONCLUSION 29 5.1 Discussion 29 5.1.1 Effects of culture medium on shoot regeneration 29 5.1.2 Effects of BAP and Kinetin on shoot regeneration 29 5.1.3 Effects of organic compounds on shoot multiplication 31 5.2 Conclusion 33 REFERRENCES 35 APPENDIX 42 vi LIST OF FIGURES Figure 1: Process of in vitro method applied in Anoectochilus setaceus Lindl conservation and propagation 12 Figure 2: Shoot regeneration rate in culture medium formula 16 Figure 3: Change in length of newly regenerated shoots in culture mediums 17 Figure Shoot regeneration rate of explants distributed in medium with different BAP concentrations 19 Figure Change in length newly regenerated shoot distributed in medium with different BAP concentrations 20 Figure Shoot regeneration rate distributed in medium with different Kinetin concentrations 22 Figure Change in length of regenerated shoot distributed in medium with different Kinetin concentrations 23 Figure Shoot multiplication rate based in organic compounds 26 Figure Change in length of multiplied shoot based on organic compounds 28 LIST OF TABLES Table Medium formulas 13 Table 2: Effect of BAP concentration on shoot regeneration 14 Table 3: Effect of Kinetin concentration on shoot regeneration 14 Table 4: Composition of multiplication medium 15 Table 5: Shoot regeneration rate in culture medium formula 16 Table 6: Average shoot length in culture medium formula 17 Table Shoot regeneration rate of explants distributed in medium with different BAP concentrations 19 Table Average regenerated shoot length of explants distributed in medium with different BAP concentrations 20 Table Shoot regeneration rate distributed in medium with different Kinetin concentrations 21 Table 10 Average regenerated shoot length distributed in medium with different Kinetin concentrations 23 Table 11 Shoot multiplication rate based in organic compounds 25 Table 12 Average regenerated shoot length based on organic compounds 27 viii PART I: INTRODUCTION 1.1 Research rationale On the edge of the sixth great extinction in history, awareness and efforts to hold back the biodiversity are higher and higher, as it is believed to be the reason for mankind's activity and the population explodes Biodiversity degradation causes extinction, threatening the extinction of more than 12.5% of flora species in the world (Walter & Gillett, 1998) Therefore, it is certainly devising to conserve the biodiversity in various approaches to optimize the chance of preserving and protecting the precious and rare species, which includes assisting to migrate to a new environment with a buffered site for challenges-adaptation (Swarts & Dixon, 2009) In the wild, the size of orchids population often declines alarmingly (Solleveld, 2017) Although orchids are the largest family of flowering plants with almost 25,000 species (Cribb et al., 2003; Dressler, 1981; Fay & Chase, 2009; Mabberley, 1997), Orchidaceae, more than any other plant family, have a high proportion of threatened genera, with most containing threatened species (Swarts & Dixon, 2009) They are exposed to the extrinsic threads from the over collecting and habitat change from human beings, as well as intrinsic (Swarts & Dixon, 2009) Besides being threatened from the outside, they, themselves, are also vulnerable, namely the complicated cooperation with mycorrhizal and the epiphytes, lithophytes characteristics Mycorrhizal is a kind of fungi that is usually helpful for orchids, as they are in a symbiotic relationship (Dearnaley, 2007), however, an imbalance, which usually occurs in a climate change context, can thrive an orchid into danger {Formatting Citation} Other than that, their ancient characteristics of epiphytes and lithophytes are now can be the cons for them, due to the global warming hit, plants tend to elevate their altitude and with these characteristics, orchids might be in trouble of re-immigrant (Swarts & Dixon, 2009) Anoectochilus setaceus Lindl belongs to the orchid family Orchidaceae It is well known for its value in horticultural, medicinal, and food usage (Fay, 2015; Gale et al., 2018) Due to the high revenue and medicinal benefits it brings, A.setaceus Lindl in Vietnam is overexploited in the wild, leading to exhaustion (Bân, 2005) In 2007, in the Decree No 32/2006 / CP and the Vietnam Red Book, A.setaceus Lindl were classified into group IA (a, c, d) a group of endangered forest plants (EN), and a group of plants banned from commercial exploitation and trade Therefore, it is very necessary to research the propagation method and cultivating tincture in an artificial environment (in vitro), contributing to the conservation of this precious medicinal herb 1.2 Research’s objectives The objective of the study was to examine the best condition for Anoectochilus setaceus Lindl.’s stems propagation by in vitro technique Many pieces of research showed orchid seeds are better used for propagation thanks to the germination (Dutra et al., 2008; Godo et al., 2010; Park et al., 2000; Zeng et al., 2012), however, due to the seasonal aspects that stems and tops are believed as a better objective to enhance the productivity and recovery of biodiversity In this study, the most suitable medium formula for A setaceus Lindl was examined based on the monitoring of shoot regeneration rate and length Other than PART V: DISCUSSION AND CONCLUSION 5.1 Discussion 5.1.1 Effects of culture medium on shoot regeneration It was indicated in that study that MS was a better candidate for the most suitable basal formula than N6, either half- or full-strength It is believed that shoots possibility’s performance depends on the salt composition medium, which MS contains more than N6 (Kriswanto et al., 2017) It can be seen from Table and Figure 2; half strength of each basic formula generated more shoots than full strength formula ½ MS showed a better result in improving the number of shoots than MS, same story was told when it comes to N6 formula For this reason, it is recommended to use only half amount of each composition in each basic studied medium This cut not only brings a better performance of cultivation, but it is also a cautious saving of resources, as well as economic benefit According to Figure and Figure 3, it proved that ½ MS was the most outstanding formula in both criteria of shoot regeneration Thereby, ½ MS was used for the following experiments in this study and highly recommended for propagating A.setaceus Lindl in vitro condition 5.1.2 Effects of BAP and Kinetin on shoot regeneration 5.1.2.1 Effects of BAP on shoot regeneration In this study, the concentration of BAP in medium was varied from mg/L (control) to 0.8 mg/L, with the final result of the best concentration was 0.8 mg/L According to Table and Figure 4, between the studied range, the more BAP added into the cultivate medium, the better results were in both tracking criteria 29 Furthermore, in Figure 4, there is a point in Week 4, after which the rate changed less rapidly It was believed to be the begining of stationary phase in shoot regeneration Ding et al., 2013 claimed that the period from Week to Week was one of the main developmental stages during in vitro cultivation of orchid, named protocorm-like bodies formation These protocorm-like bodies then develop into the vegetative stage of a plant that shoots are less likely to be produced In conclude from Figure and Figure 5, it showed clearly a significant impact of BAP in increasing the number of new shoots, as was proved by the fact that the performance of medium with BAP in any concentration was better than the control medium’s result Although 0.8 mg/L BAP contributed to the accomplishment of the longest shoot, as can be seen in Figure 5, the gap between the highest, second highest or even third highest regeneration rate was not significant For that reason, the use of less BAP in cultivating medium does not seem to drop the productivity of shoot elongation However, with the purpose of improve the shoot regeneration in both criteria, it is still recommended to use 0.8 mg/L BAP for the best performance 5.1.2.2 Effects of Kinetin on shoot regeneration It can be seen in Figure that the development of regeneration rates of all medium that including Kinetin were higher than the one without (control medium) This result proved the effectiveness of Kinetin as a growth regulator on shoot regeneration rate In this criterion, the efficiency of 0.8 mg/L Kinetin was higher than of mg/L For that reason, the amount of Kinetin was not associated with the number of newly regenerated shoots Timing, in fact, was a considerable factor In 30 Figure 7, 0.8 mg/L, 0.4 mg/L, 0.2 mg/L worked better than mg/L with elongating shoot length from Week to Week It is recommended to have further study about the timing associated with Kinetin’s efficiency to determine the best gathering time Comparing the efficiency of BAP and Kinetin, Figure and Figure 6, Figure and Figure showed that BAP seemed to be a better growth stimulator for A.setaceus Lindl in vitro conservation and propagation 5.1.3 Effects of organic compounds on shoot multiplication The result of this study showed that the association of all the compositions brought that greatest benefit to the multiplication stage of A.setaceus Lindl in vitro conservation and propagation The use of 100 ml/L Coconut water + 10g/L Potato + 10g/L Carrot is highly recommended as an organic compound 5.1.4 Implementation of in vitro propagation in A.setaceus Lindl conservation and management Studies in generating a potential population by artificial propagation are recommended for future orchid conservation (Wraith et al., 2020) However, the number of A.setaceus Lindl is believed to continuingly decline if there is not a practical implimentation This study can only fill in the current gap between theory and practice with the application of it in practical use Fay, 2018 advocated that the conservation of orchid in a sterilized, isolated condition, without the association with fungal and pollinators can only mean to the restoration of the species Hence, the techniques of in vitro must come along to support in situ conservation of A.setaceus Lindl In order to rehabitat the orchid back into the 31 wild, the collaborations between research institute and conservationist are highly recommended to acquire (Pedersen et al., 2018) Evaluating the transplantation location towards self-sustaining population asks fellow scientists to concern about threats, land regions, conservation priorities in the region, mycorrhizal and availability of pollinators (Wraith et al., 2020) Providing a spatial map with multicriteria analysis (MCA), regarding aforementioned criteria, defines the potential distribution for transplantation of A.setaceus Lindl Protecting and managing threatened orchids from illegal collection, harvesting and logging are another priority that demands the dedication of government and policy makers (Wraith et al., 2020) This chain manages the biodiversity and secures the effectiveness of conservation from being disturbed by human activities A proper governance can coordinate these impacts to minimum, for instance, the encouragement of government for ecotourism rather than traditional tourism can promisingly limit the influence of human beings to the habitat (N D Swarts & Dixon, 2009) Moreover, it is important for the local collectors to aware the exploitation and endangered status of A.setaceus Lindl Therefore, raising education and awareness programs must be conducted This recommendation might also change a livelihood for people who used to depend on forestry resources, and orchid (Wraith et al., 2020) Most importantly, the conservation policy must be stricter, starting with updating the profile of A.setaceus Lindl conservation priority to date (Wraith et al., 2020), as the last edition of Vietnam Red Book is from 2007 Vietnam belongs 32 to the most exploitation and trading orchid countries (Wraith et al., 2020), therefore the changes are more terrific that needs to be up to date Commented [TT2]: 5.2 Conclusion The implementation of in vitro technique into the conservation of A.setaceus Lindl promises to bring a high productivity of propagation, and a buffered site for challenge adaptation for rehabitatant The result of this study is only used for the conservation of A.setaceus Lindl., any commercial application from this study is not recommended The most suitable basic medium for conservation and propagation A.setaceus Lindl in vitro is ½ MS Between studied growth regulators, BAP is highly recommended in use rather than Kinetin, thanks to its excellent performance with both tracking criteria, and to avoid the complicated use of Kinetin due to its dependent factor - timing When it comes to shoot multiplication in medium containing organic compounds, it is highly recommended to use 100 ml/L Coconut water + 10g/L Potato + 10g/L Carrot for the best record In a nutshell, the most efficiency medium for shoot regeneration of Anoectochilus setaceus Lindl., based on studied factors, is ½ MS+ 0.8 mg/L BAP + 100 ml/L Coconut water + 10g/L Potato + 10g/L Carrot In order to meet the technical requirements of this study and the final aim of recovering Anoectochilus setaceus Lindl., it is recommended the collarboration between the conservationist work force, research institues and the goverment The conservation of this precious species can only be efficient with a proper management which prioritizes to limit the impacts of human in the natural habitat In order to minimize the influences, it 33 is important to raise the awareness of people on the current critical conservation status and change the livelihood of people who used to depend on the forest resources 34 REFERRENCES Vietnamese: Bân, N T (2005) Danh lục loài thực vật Việt Nam, tập Nhà Xuất Bản Nông Nghiệp, Hà Nội Phê, P., Thành, N., Hưng, V (2010) Đặc điểm hình thái, phân bố loài lan Kim tuyến Anoectochilus setaceus blume Vườn Quốc gia Tam Đảo, tỉnh Vĩnh 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N-(2furfurylamino)-1-H- purine-6-amine (kinetin), and 6-(4-hydroxy-3-methyl-trans2-butanylamino) purine (zeatin) Zeatin and 2-iP are natural cytokinins, while BA and kinetin are man-made cytokinins Generally,... Effect of Kinetin concentration on shoot regeneration Formula Kinetin (mg/l) Kinetin ( 1) (Control) Kinetin ( 2) 0.2 Kinetin ( 3) 0.4 Kinetin ( 4) 0.8 Kinetin ( 5) 1.0 Monitoring criteria: Rate of regenerated... medium and objective explants in vitro toward the conservation and propagation of Anoectochilus setaceus Lindl can be identified 1.3 Significance of the study In vitro is the method referring the