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 : Environmental Science and Management Faculty : Advanced Education Program Batch : 2017 – 2021 Thai Nguyen, 06/2021 Luan van 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. Nguyen Tien Dung Supervisor Dr. 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. 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.
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.
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. conservation priority to date. ii Luan van Anoectochilus setaceus Lindl, in vitro propagation, Keywords conservation Number of pages Date of submission June, 2021 iii Luan van 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. 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. Luan van TABLE OF CONTENTS ACKNOWLEDGEMENT.
iv TABLE OF CONTENTS. v LIST OF FIGURES .vii LIST OF TABLES .viii PART I: INTRODUCTION. Significance of the study. Scope and Limitations.
Orchid conservation status. Anoectochilus setaceus Lindl. Effects of basal culture medium on shoot regeneration. Disinfection of sample.
Effects of 6-Benzylaminopurine (BAP) and Kinetin on shoot regeneration. Effect of BAP on shoot regeneration. Effects of Kinetin in shoot regeneration. Effects of some organic compounds (coconut water, potatoes, carrots) on multiplication of cultured samples.
Effects of culture medium formula on shoot regeneration. Effects of culture medium formula on shoot regeneration rate. Effects of culture medium on average regenerated shoot length. Effects of 6-Benzylaminopurine (BAP) and Kinetin in shoot regeneration stage.
Effects of BAP on shoot regeneration stage. Effects of Kinetin on shoot regeneration stage. Effects of some organic compounds (coconut water, potatoes, mashed carrot) on shoot multiplication stage. Effects of some organic compounds (coconut water, potatoes, mashed carrot) on shoot multiplication rate.
Effects of some organic compounds (coconut water, potatoes, mashed carrot) on multiplied shoot length. 27 PART V: DISCUSSION AND CONCLUSION. Effects of culture medium on shoot regeneration. Effects of BAP and Kinetin on shoot regeneration.
Effects of organic compounds on shoot multiplication. 42 vi Luan van 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. Shoot regeneration rate of explants distributed in medium with different BAP concentrations. Change in length newly regenerated shoot distributed in medium with different BAP concentrations. Shoot regeneration rate distributed in medium with different Kinetin concentrations.
Change in length of regenerated shoot distributed in medium with different Kinetin concentrations. Shoot multiplication rate based in organic compounds. Change in length of multiplied shoot based on organic compounds. 28 Luan van LIST OF TABLES Table 1.
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. Shoot regeneration rate of explants distributed in medium with different BAP concentrations. Average regenerated shoot length of explants distributed in medium with different BAP concentrations. Shoot regeneration rate distributed in medium with different Kinetin concentrations.
Average regenerated shoot length distributed in medium with different Kinetin concentrations. Shoot multiplication rate based in organic compounds. Average regenerated shoot length based on organic compounds. 27 viii Luan van PART I: INTRODUCTION 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 1 Luan van 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.
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.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. was examined based on the monitoring of shoot regeneration rate and length. Other than 2 Luan van that, with the inheritance of previous studies by Biotech laboratory in Thai Nguyen University of Agriculture and Forestry (TUAF), the effects of different growth regulators (BAP and Kinetin) concentration on the shoot regeneration rate and the quality of regenerated shoots were determined.
After shoot regenerating, shoots were then allocated in a different medium with organic compounds differ by composition, to tracking the shoot multiplication. Through this study, the most suitable culture medium and objective explants in vitro toward the conservation and propagation of Anoectochilus setaceus Lindl can be identified. Significance of the study In vitro is the method referring the experiments outside of a living organism which is highlight to preserve the organism. It provides all the supplement that a tissue of an explant needs to nurture in lab condition.
In vitro regards a buffered site for challenge adaptation of such vulnerable species before habitant into the wild. This study, implementing the advantages of in vitro technique into the propagation of A. helps preserving this precious orchid and maintaining biodiversity. Scope and Limitations Several limitations of the experiment could lead to different results.
During the medium preparation, the agar was not distributed into the mediums equally, leads to some of the mediums were only semi-fluid but not semi-solid, so that it may cause the falling of explants as the lack of firm support. 3 Luan van PART II. Orchid conservation status Anthropogenic factors of wild orchids collecting and land clearance directly drive orchids into rarity (Cribb et al. Overcollection, usually illegal and undocumented is the extraordinary deprivation of orchids diversity (IUCN, 1999).
Exploiting the ecosystem by hunting can also thrive the food chain into a mess which can imbalance the grazing pressure on orchids and make them more exposed to damaged (Swarts, 2007). Other extrinsic threats from human impacts are the unsustainable acceleration of civilization, industrialization, etc. Thus, kick-on effects, disease, pests, intense fire, salinization, and desertification have the potential condition to spread out and challenge the natural habitat of orchid, as well as orchid conservation (Sahagian, 2000). In the context of habitat changes, a number of significant risks may be occurred by the association of the aforementioned factor (Brundrett, 2006), an orchid is more likely to be associated by mycorrhizal and then recorded as the first reducing the abundancy in the disturbed ecosystem (see (Cribb et al., 2003) for case studies of specialized attributes in orchids).
Under the climatic change on a global scale recently, epiphytes and lithophytes orchid has been vulnerable due to the elevation of medium objects’ distribution. Furthermore, the pollinator of orchid depending on a specialized seed dispersal agent, can also transmigrate or be extirpated due to climate change, therefore, can enhance the extinction level of orchid (Dixon, 1991).