Nghiên cứu một số cơ sở khoa học nhằm bảo tồn, phát triển và sử dụng bền vững một số loài trong chỉ kim tuyến (anoectochilus blume) ở việt nam tt tiếng anh

MINIS TRY OF EDUCATION AND TRAINING VIET NAM ACADEMY OF SCIENCE AND TECHNOLOGY INSITITUTE OF ECOLOGY AND BIOLOGICAL RESOURCES PHAN XUAN BINH MINH RESEARCH SOME SCIENCE BASIS FOR CONSERVATION, DEVELOPMENT AND USE SUSTAIN SOME SPECIES OF Anoectochilus Blume GENUS OF VIETNAM Major Code : Plants : 42 01 11 SUMMARY OF BIOLOGY DOCTORAL THESIS HA NOI - 2019 Thesis completed in: Insititute of Ecology and Biological Resources -Viet Nam Academy of Science and Technology Super visor 1: Assoc Prof Dr Tran Minh Hoi Super visor 2: Dr Pham Huong Son Reviewer 1: … Reviewer 2: … Reviewer 3: … Thesis will be protected before the doctoral thesis evaluation board of the Institute, meeting at Institute of Ecology and Biological Resources -Viet Nam Academy of Science and Technology at day month year Thesis save at: - Library of Institute of Ecology and Biological Resources - National library of Viet Nam 2 LIST OF WORKS DISCLOSURE OF THE AUTHOR RELATED TO THE THESIS Phan Xuan Binh Minh, Pham Huong Son, Tran Minh Hoi, Nguyen Thi Van, (2015), Propagation Solutions for Conservation and Development of Anoectochilus lylei Rolfe ex Downie, Proceeding of the th Nationnal Scientific Conference on Ecology and Biological Resources, Publisher of Natural Science and Technology : 695 – 699 Pham Huong Sơn, Nguyen Thị Lai, Phan Xuan Binh Minh, (2015), Isolation and simultaneous quantification ofadenosine, narcissin and roseoside in Anoectochilus roxburghii(Wall.) Wall ex Lindl, Journal of Chemical, Biological and Physical Sciences (4): 4002 – 4007 Tran Minh Hoi, Tran Van Thai, Chu Thi Thu Ha, Ha Thi Van Anh, Phan Xuan Binh Minh, (2016), Flavonoids from Anoectochilus annamensis and their Anti-inflammatory Activity, Natural Product Communications 11(5): 613 - 614 Phan Xuan Binh Minh, Pham Huong Son, (2016), Distridution, Habitat of Five Species of the Genus AnoectochilusBlume in Vietnam, Proceedings The 12th Asia Pacific Orchid Conference: 110- 116 Phan Xuan Binh Minh, Pham Huong Son, Tran Minh Hoi, (2017), Liquid Medium for Efficient Micropropagation of Anoectochilus annamensisAver., Proceeding of the th Nationnal Scientific Conference on Ecology and Biological Resources, Publisher of Natural Science and Technology : 1756- 1762 Phan Xuan Binh Minh, Bui Thị Thanh Phuong, Pham Huong Son, Nguyen Thị Phuong Lan, Vu Thi Thao, (2018), The effects of linght emitting diode lighting on growth and development of A annanesis and A roxburghii in vitro cultured shoots, Journal of Biology, 40(1): 32- 38 DOI: 10.15625/0866-7160/v40nl 10636 INTRODUCTION 1.The urgency of the thesis topic Anoectochilus Blume genus belongs to Orchidaceae Juss There are about 50 species in the world In Viet Nam, according to Nguyen Tien Ban al et (2005) there are 12 species of Anoectochilus Blume, in which species are named in the red book, these are: Anoectochilus acalcaratus Aver., Anoectochilus calcareus Aver., A chapaensis Gagnep., A setaceus Blume, A tridentatus Seidenf ex Aver.) According to Averyanov LV (2008), Anoectochilus Blume in Vietnam accounts for out of 30 species of tropical Asia This genus is included in the List of endangered and rare forest plants (Group IA) of Decree No 32/2006 / ND-CP dated March 30, 2006 of the Government, prohibited for exploitation and only used for commercial purposes Like Vietnam, the species in the world of Anoectochilus genus are also collected in the wild Many species have scientific significance (diversity of genetic resources, endemism) and high use value (especially for medicine) such as: Anoectochilus koshunensis, Anoectochilus sandvicensis, Anoectochilus zhejiangensis… are at risk of extinction Therefore, it is necessary to find solutions to conserve and sustainably develop some species, especially valuable ones Research objectives of the thesis topic Studying on morphological characteristics, distribution, status and chemical composition and propagation method and cultivating some species of Anoectochilus Blume genus as a basis for proposing conservation solutions, sustainable development and use of species belonging to Anoectochilus Blume in Vietnam Scientific and practical significance of the thesis topic 3.1 Scientific Providing, supplementing new scientific data on Anoectochilus Blume genus in Vietnam such as the distribution of species, active ingredient of species 3.2 Practical significance It is a scientific basis for the sustainable development of some species in the Anoectochilus Blume genus in Viet Nam New points of the thesis - This is a relatively complete and systematic scientific research project (biological, ecological, distribution characteristics) of valuable Anoectochilus Blume species in Vietnam (A annamensis, A calareus, A elwesii, A lylei and A setaceus) - Studying on the chemical composition of species A setaceus and Anoectochilus annamensis and determine: + compounds from A Setaceus, including newly added compounds, adensine and reoside + compounds from A annamensis, including new compound 4’, 5dihydroxy-3,3’, 7- trimethoxyflavone 4’-O-α-L-rhamnopyranosyl- (1→6)-β-Dglucopyranoside - The study identified scientific bases for successful propagating of A annamensis, A lylei and A setaceus in vitro culture methods from seeds and buds - Proposing some initial solutions, feasible to propagate , cultivating for conservation in relocation conditions (ex situ) combined with conservation in place (in situ) of species A annamensis, A lylei and A setaceus - Investigating, evaluating and collecting some information on the current status of exploitation, trading and consumption of some species Anoectocilus at National Parks, nature reserve and some areas have forests in some localities - Base on the research results, the system has proposed solutions for effective conservation of species belonging to the Anoectochilus genus The layout of the thesis The entire thesis consists of 156 pages, 20 drawings, 49 pictures including parts: - Introduction: pages - Chapter 1: Overview of research issues: 30 pages - Chapter 2: Subjects, content and research methods: pages - Chapter 3: Research results and discussion: 94 pages - Conclusions and recommendations: pages - References: 17 pages CHAPTER 1: OVERVIEW OF RESEARCH ISSUES 1.1 Overview of Anoetochilus Blume Anoectochilus belongs to orchid which includes about 40-50 different small terrestrial herbs from the Himalayas in India to the mountains of Southeast Asia, southern China, Japan, Indonesia, and some Pacific Islands Most of the Anoectochilus are small herbaceous species, growing on the soil, leaves of some species with yellow-or-pink veins Flowers are small but the lower lip is large in size compared to flowers and divided into two clear lobes Anoectochilus is known as a precious medicinal herb that fosters health, snakebite treatment and liver diseases Local people use Anoectochilus in the forest as a natural gift to promote health 1.2 The situation of research on Anoectochilus Blume in the world - The results of research on Anoectochilus in the world are mainly carried out in China and there are also countries like India, Thailand and other Southeast Asian countries The studies on Anoectochilus come in three main directions: Reseach on plants (species composition, distribution area, reality ) The researches on main uses and medicinal values, studies on propatating and cultivating solutions - The results of the study show that the Anoectochilus genus with about 30 to 50 species in the world is distributed throughout continents of Asia, Australia, Papua New Guinea and some Pacific islands In which China has about 13 species, India has about 16 species and Southeast Asian countries has about 20 species - The results of the study on the use of Anoectochilus species: According to traditional medicine Anoectochilus is a very valuable medicine in the herb shops, the medicinal plant is cool and sweet, Antipyretic, cooling blood, good for lung, dissolving cancer, fostering health, healing organs such as hot liver, lung disease, Haemorrhage, cough, chest pain, splenalgic, sore throat, Hypertension, retarded children, kidney failure - Medicinal Value According to modern medicine, there are many research results indicating that purl species contain active substances such as: flavonoid, Amino acids have antibacterial, anti-aging, hypotension, diabetes and paralyzes the mechanism of strange cell activity There are currently products and active ingredients of some Anoectochilus species such as: A setaceus, A formosanus… were commercial The results of the study on on propatating and cultivating: The results of this study began in the 80s and 90s in China The results showe that the propagation method is mainly in vitro propagation from seeds in culture medium with microbial supplement to support the growth and development of Anoectochilus In addition, recent studies have used liquid propagation culture medium systems and LED lighting systems The cultivating method has two methods: Cultivating in the greenhouse and cultivating under forest canopy 1.3 The situation of research on Anoectochilus Blume in Vietnam The results of research, survey, inventory of species composition have been implemented by Pham Hoang Ho, Nguyen Thien Tịch, Nguyen Tien Ban and Averyanov L since the 90s of the last century The research results on chemical composition and medicinal value of Anoectochilus are very limited Vu Cong Luan et al (2005) published the results of propagation and quantitative β- sitosterrol, an active ingredient used to treat cardiovascular diseases in culture medium plants After that, author Do Thi Gam et al (2017) also extracted and quantified Flavonoid total over three species of Anoecochilus (A setaceus, A annamensis, A lylei) - New propagating studies have focused on in vitro propagation from some particular buds (A setaceus, A formossanus) CHAPTER 2: SUBJECTS, LOCATION AND RESEARCH METHODS 2.1 Research subjects The object of the study is the species of genus (Anoectochilus Blume), distributed in nature in Vietnam Surveying, collecting samples, determining the distribution area and assessment of species exploitation reserves is based on survey results, interview local people, management staff at national parks, nature reserves and other forests 2.2 Research location 18 survey points: Ba Be national park, Xuan Son national park, Tam Dao national park, Phong Nha -Ke Bang national park, Bach Ma national park, Chư Yang Sin national park, Khau Ca nature reserve, Hoang Lien - Van ban nature reserve, Than Sa Phuong Hoang nature reserve, Xuan Lien nature reserve, Bac Huonng Hoa nature reserve, huyen Hoang Su Phi – Ha Giang, huyen Bac Me – Ha Giang, huyen Tra Linh - Cao Bang, huyen Sa Pa- Lao Cai, huyen Ky SonNghe An, huyen Nam Tra Mi - Quang Nam huyen Bao Loc - Lam Đong The studies of extraction and distribution of active ingredients were conducted in the Department of Biological Activities - Marine Biochemistry Institute, Vietnam Academy of Science and Technology The site for cultivating and propagating research is carried out at: Tissue culture laboratory and Nursery Center of Experimental Biology – national center for Technology progress Location of growing area is implemented at: Biopharma Company, Hoa Binh 2.3 The main research content of the thesis Morphological characteristics Anoectochilus in VietNam and classification of species on Determination of the active ingredient in two species of loài A setaceus A annamensis Studying and investigating the actual situation of exploitation and trade of species belonging to the genus Anoectochilus Then giving management and propaganda solutions for local people to understand the value of Anoectochilus species and protect them Proposing solutions to conserve and sustainably develop rare species belonging to the Anoectochilus + Solution for management: Bringing noectochilus species to the list of medicinal plants that need to be protected, proposing reasonable conservation areas and propaganding in the community + Technical solution: Research on propagation techniques by cultivating method in vitro, cultivates Anoectochilus species A annamensis, A setaceus, A lylei serving conservation and sustainable development 2.4 Research methods 2.4.1 Methods of data collection in the field Field survey and samples collection following plant research method of Nguyen Nghia Thin (2007) Interview method and Rural Rapid Appraisal (RRA) 2.4.2 Method of classification and identifying species names - Using expert methods to and document of Pham Hoang Ho (2000), Nguyen Thien Tich (2001), Nguyen Tien Ban (2005) and Averyanov L.V (2008) to classify identifying species by morphology methods 2.4.3 Methods of determining chemical composition - Total methanol extraction method, The extract with methanol (3L x4 times) in a sonic bath for 30 - Isolation method: Isolate pure substances by common chromatographic methods such as normal phase column chromatography (normal silica gel), reverse phase (RP18, YMC ODS), use ion exchange resins (Dianion HP20) or particle size adsorption (Sephadex LH20) - Methods of determining chemical structure: Determining the structure of isolated substances by modern spectroscopic methods including infrared absorption (IR), ultraviolet (UV), one- and two-dimensional magnetic resonance spectra (1D and 2D-NMR), normal (MS) and high resolution (HR MS) mass spectra 2.4.4 Method of propagation by in vitro culture Method of propagation by in vitro from buds: Study appropriate propagation conditions when using dormant buds as material culture for starting materials Methods of propagation by in vitro from seed: Study the selection of propagating conditions suitable for shoot proliferation and root formation Methods of selection of suitable culture conditions: Study of modern in vitro culture techniques such as: Liquid culture culture, using lighting system LED 2.4.5 Method of cultivating ex vitro - Method of cultivating in greenhouse: Study suitable conditions when cultivating plants in vitro in greenhouse - Method of cultivating under forest canopy: Seasonal research, pests, How to prevent when planting under the forest canopy 2.4.6 Processing methods and statistics Methods of processing and analyzing data on software Iristat 5.0 CHAPTER 3: RESEARCH RESULTS AND DISCUSSION 3.1 Morphological characteristics, distribution of Anoectochilus in Vietnam 3.1.1 Morphological characteristics The genus Anoectochilus are terrestrial, perennial, deciduous, sympodial herbs with a creeping, above-ground rhizome with wiry roots that look woolly The leaves black or gray-black, rarely dark gray-brown with green tint, commonly dense reticulated with net of silvery-white nerves The flowers are relatively large, hairy, velvety, resupinate and arranged in a short spike Mesochile flangeswith at least 1–2 long linear fringes 3–5mm long The dorsal sepal and petals overlap to form a hood over the column with the lateral sepals spreading apart from each other The labellum is relatively large with two sections - an upper "epichile" and lower "hypochile" separated by a narrow section The hypochile has a cylindershaped spur containing two large glands and is joined to the epichile with a "claw" that has spreading teeth or a long fringe The fruit is a hairy capsule containing a large number of winged seed Plants regenerate in nature from buds and seeds During the survey period from 2012-2017 at national parks, natural reservé and points have forests in many different provinces, The team collected and identified species of the genus Anoectochilus including: A annamensis Aver.; A calcareus Aver.; A elwesii C.B Clarke ex Hook.; A lylei Rolfe ex Downie0(A setaceus Wall ex Lindl 3.1.2 Distribution of species belonging to the genus Anoectochilus Survey of national parks, natural reserves and forested areas in geographical regions and typical ecosystems from North to South, most of the Anoectochilus species in Vietnam are distributed in Tropical broadleaf evergreen 10 ARW10.1 3.5-Dihydroxy-3’4’-7-trimethocy flavone ARW11.4 Adenosine-5' methyl sunpua ARW12.2 (6S,9S)-Roseoside ARW18.7 Stigmasterol ARW13.7 mix Benzyl-β-Dglucopyranoside and methylarbutin 3.2.2 Isolation and determination of chemical structure of substances in A annamensis From the MeOH extract of the A annamensis, compounds have been isolated as shown in Table 3.3 13 Table 3.3: List of substances isolated from the A annamensis numerical order sign Structure AAH9.10 Molecular formula, molecular weight note C18H16O7 M = 344 - C28H32O16 M = 624 - C30H36O16 M = 652 Chất 5,4'-dihydroxy-3,7,3'trimethoxyflavonone AAE14.18 (trùng với ARW3.5) Isorhamnetin 3-Orutinoside AAE14.13 14 5-Hydroxy-3,7,3'trimethoxyflavone 4'-Oα-L-rhamnopyranosyl(1→6)-β-Dglucopyranoside AAH8.2 C28H40O M = 392 - C39H68O5 M = 616 - C24H38O4 M = 390 - Ergosta-4,6,8(14),22tetraen-3-one (Ergone) AAH10.9 (2S)-2,3-O-dioctadeca 9Z,12Z-dienoyl-glycerol AAH8.15 Di-(2-ethylhexyl) phthalate (DEHP) 3.3 Status quo of exploitation, use and trade of species belonging to the genus Anoectochilus Blume 3.3.1 Status quo of exploitation In the last 10 years, the demand for purchasing medicinal materials in general and species of Anoectochilus has increased rapidly In 11 surveyed areas, there are bosses buying NTFP and 100% of them have purchased Anoectochilus According to the local people in these 11 regions, the exploitation of Anoectochilus began in the late 90s, developed strongly in the early 2000s when 15 Chinese bosses ordered to buy Until now, the exploitation and purchasing continues to take place, but the number and scale are smaller due to the depleted natural resources 3.3.2 Status quo of use and trade and management of Anoectochilus Blume species locally At the 18 sample collection sites, people have collected and traded species belonging to the Anoectochilus genus In researching and surveying about understanding and value of using species belonging to Anoectochilus genus, The research team chose National Parks, Natural Reserves, other forest sites are known that almost all exploited Anoectochilus is for sale at a rate of less than 10% The reason is that most people as well as managers not know or know very little about the value of genetic resources as well as the use value of Anoectochilus According to survey data from 165 local people participating in exploitation and and 16 NTFP traders at 11 survey points The results show that the exploitation and purchasing of Anoectochilus Blume species still occurs daily at many different points all over the nation, The number of exploitation days in a year depends on the weather, customs and ecological characteristics of the species Local people who go to the forest much should understand the rules to exploit Through research, it is known that due to being eradicated for many consecutive years, the natural source of Anoectochilus exhausted gradually, current exploitation is only 1/2 or 1/3 compared to 10 years ago Currently, Anoectochilus is only very deep in the forest or rugged terrain Therefore, the participants in the exploitation are also narrowed down and there is no more participation of children and women as before, but the exploitation area was expanded At some areas like Tam Dao National Park, Phong Nha Ke Bang National Park , Hoang Lien-Văn Bàn nature reserve or South Trà My – Quảng Nam, people not only exploit forests near their houses They formed groups of 2-3 men to go to the forest after 3-5 days they return, the target of their exploitation is ornamental plants and medicinal herbs including Anoectochilus Blume 3.4 Solutions to preserve and develop Anoectochilus 3.4.1 Solution of propagating and cultivating three species of Anoectochilus Blume ( A annamensis, A lylei ,A setaceus ) 16 In the situation of endangered Anoectochilus species due to various causes, the main cause is the eradicating and uncontrolled trade Propagating and cultivating are the most effective conservation and development solutions 3.4.1.1 Solution to propagate three species of Anoectochilus by using in vitro culture method For Orchid family in general and Anoectochilus genus in particular, propagation by in vitro culture method, especially in vitro culture from seeds is an effective solution for many countries around the world such as: Japan, China, Thailand used In in vitro propagation there are three main stages: Shoot induction, Shoot growth, Roor growth a Shoot induction In research, there are two main contents to find mineral culture medium formulas, growth regulator and appropriate concentrations for each research object Influence of mineral culture medium and growth regulator on the budding ability of the A annamensis, A lylei Research results show that the culture medium is suitable for regeneration of A annamensis is shoots cultured on MS medium supplemented 1.0 mg/l TDZ culture medium of A lyle grown best in MS medium supplement with 1,5mg/l BAP - Influence of mineral culture medium and growth regulators on germination ability of A annamensis, A lylei, A setaceus Research results of the effects of mineral medium and growth regulators on the germination ability of three species of Anoectochilus Blume (A annamensis, A lylei, A setaceus.) is shown in Table 3.8 Table 3.8: Influence of mineral medium and growth regulators on germination ability A annamensis seedlings cultured in vitro Concentration Medium TDZ (mg/l) Rate of green sprouts (%) 17 The average mass of the sprouts (mg/sprout) MS Knud HY 17.1f 0.42d 0.1 21.5e 0.67c 0.5 36.4d 0.75c 73.2a 1.42b 1.5 65.6b 1.86a 2.0 58.7c 1.93a LSD0.05 3.1 0.1 15.8f 0.46e 0.1 22.2e 0.64d 0.5 31.7d 0.83c 62.7a 1.29b 1.5 59.7b 1.56a 2.0 56.3c 1.62a LSD0.05 2.7 0.08 16.4f 0.47f 0.1 20.5e 0.72e 0.5 29.7d 0.89d 66.2a 1.35c 1.5 61.7b 1.74b 2.0 52.8c 1.87a LSD0.05 3.4 0.12 18 VW 15.8f 0.42f 0.1 19.7e 0.59e 0.5 29.2d 0.72d 57.4a 1.07c 1.5 51.8b 1.52b 2.0 48.4c 1.64a LSD0.05 2.4 0.06 Note: LSD0.05 is the smallest significant error at the permissible level of 5%0; Different letters (a, b, c ) are given in columns that represent significant differences at LSD level Research of results showed that on MS medium supplemented 1.0 mg/l TDZ is sutable for A.annamensis Seed germination (rate of Germination 73.2%; The average mass of the sprouts 1.42mmg/sprout after 16 weeks culturing) This is a pretty good result compared to the results of Yih-Juh Shiau al et (2002) in the cultivation of A formosanus seeds on MS medium supplemented 2mg BAP results after months of culture (rate of Germination 81.4%; The average mass of the sprouts 1.84mg) The same for A.lyle and A setaceus Seeds are appropriate Culturing on MS medium supplemented 1.5 mg/l BAP A.lyle after 16 weeks in culture A lyle on Culture medium MS+ 1.5mg/l BAP for best results in both indicators: (rate of Germination 64.7%, The average mass of the sprouts 1.82mmg So is Anoectochilus after 16 weeks in culture on MS medium + 1.5mg/BAP for best result: rate of Germination 75.2%, The average mass of the sprouts 1.62mg b Shoot growth Shoot growth is the period of increase in shoot multiplier, this stage needs to achieve these requirements: Creating the highest propagating (short culture time, high quantity) Homogeneous produced shoots have good growth potential, so when the trees are completed they grow root healthily and the possibility of malformations is lowest In this period, there are two research contents: 19 - Effect of Culture conditions: The result ofAnoectochilus annamensis grows best in MS Medium supplemented with 0.5mg/l TDZ after weeks in culture for mean number of shoots per explants 9.23, Shoots grow well As for A lylei after weeks in culture for mean number of shoots per explants 9.12 Using liquid medium in Shoot growth not only increases biomass, cost savings it is also a premise for large-scale seed production processes and research to create artificial seeds This method has been applied on many crop objects Research results on three different propagating conditions: Solid medium, liquidshaked (80rpm), liquid medium, supplemented with cotton to cultivate species of A annamensis, A lylei, A setaceus showed that in the liquid-shaked medium, the mean number of shoots per explants was higher than the solid medium and the cotton medium in all three Anoectochilus Blume species Liquid-shaked medium stimulates the process of shoots and roots to grow stronger and more importantly the morphology of the tree is not changed In addition to adapting to the liquidshaked medium conditions of the growing root for the tree stands up-right, the diameter is larger and the stems are harder These are also important indicators for seedlings in vitro Using liquid medium in In vitro propagation in general and medicinal plants in particular to increase weight and lower product costs have been implemented in some countries such as Japan, India these studies - Effect of lighting conditions In Shoot growth The determination of suitable culture medium and external conditions is crucial to the efficiency of Shoot growth processing In nature, most of Anoectochilus are distributed under the evergreen forest canopy with a canopy cover of 70-80% so these species are suitable for low light According to Ma ZQ et al (2010), A formosanaus is suitable for 30- µmol / m2 / s lux ligh In this study, LED illumination sources are in different wavelengths of (λ= 430-470 nm; λ= 470-510 nm; λ= 510-560 nm; λ= 560- 600 nm) with light intensily photosynthetic photon flux density (PPFD) of 40µmol/m2/s photon lighting time 12h /day, The control formula is fluorescent lamps on three objects A annamensis, A lylei, A setaceus Results after weeks of culture were obtained: A annamensis is not only influenced by environmental components, growth regulators but also influenced by lighting conditions LED light with wavelength λ = 430-470 nm has the highest ability to stimulate shoot formation (12.73 shoots / sample) higher 1.4 compared to the control But at the wavelength λ = 470 - 510 nm, it is more suitable for the development of buds A annamensis 20 and at this wavelength the leaves also grow better than the wavelength λ = 430470 nm and Other lighting levels For A lylei with LED wavelength 470 = 470510 nm in this illumination mode, all three parameters of shoots, height and average weight of shoots were higher than those of control and with other formulas Similar to A lylei, A setaceus also suitable with LED lighting mode at wavelength range λ = 470- 510 nm, in this lighting mode all three indicators, mean number of shoots per explants, mean height of shoots, mean weight of explants are all higher than other lighting modes, Especially much higher than the control formula is fluorescent (mean number of shoots per explants 12.67 higher than 1.43 compared with the control was 8.87), Other indicators such as average height or weight are also higher than the control formula and other experimental formulas The results showed that the LED lighting system has a positive influence on the growth and development of in vitro cultivating shoots of two species of Anoectochilus Both species grow well when using LED lighting system with wavelength range λ = 470- 510 nm, however, for A annamensis, the wavelength range λ = 430- 470nm helps stimulate more shoot production c Root growth - Effect of concentration of growth regulator Study the effects of NAA and IBA at concentrations 0.1; 0.5; 1; 1.5; mg / l The results showed that for A annamensis at 0.5 mg / l for both auxin types, the rooting rate as well as the average number of roots and root length were better than those at the concentration other But giving the best results is still in the formula 0.5mg / l IBA with the indicators (frequency of shoots forming roots 93.33%, mean number of roots per shoot 2.64; mean leghth of roots 11.60mm) So the most suitable concentration for A lylei is NAA at a concentration of 0.5mg / l with frequency of shoots forming roots 89.72%, mean number of roots per shoot 2.78, mean leghth of roots 10.76mm - Effect of culture Medium conditions From the positive results when using liquid medium in the Shoot proliferation of three species of Anoectochilus Further research was carried out during the root formation on medium formulas MS supplemented 0.5mg/l IBA and MS medium supplemented 0.5mg/l NAA in different mediums: Solid medium ( supplemented 8g agar/l), cotton medium, liquid medium and Shaked at 60 rpm for three species A annamensis, A lylei, A setaceus results after weeks of culture The above results show that for all three types of Anoectochilus Blume of 21 A annamensis, A lylei, A setaceus of the liquid-shaked medium, the best results are obtained Liquid-shaked medium stimulated the formation and development of roots better than static liquid environment and solid environment, moreover the liquid-shaked environment also helped the root system to develop more evenly, more feathers on the roots help the plants stand steadily 3.4.1.2 Solutions for Culturing Anoectochilus a Culturing in greenhouse conditions The stage of putting in vitro plants into nursery is a very important, determine the applicability of the entire culture process in vitro to production practices This is also the period of many difficulties for transferring plants from test tubes to nurseries resulting in sudden changes in living conditions from artificial environments to natural conditions with many difficulties to control natural factors such as: light, temperature, humidity… tested on organic mulch and coco peat Tri Bat with 25% and 50% earthworm fertilizer extract after months of growing in the condition of net houses with average temperature from 20-30oC, the average lighting intensity is 40-50 µmol / m2 / s After 16 weeks of culture, the result is three species of loài A annamensis, A lylei, A setaceus which grow and develop well when plant on organic mulch and coconut mulch with 25% of earthworm fertilizer Continuing culture in the above conditions after 10-12 months for harvest b, Culturing in the wild - Effect of planting season In addition to cultivating in greenhouses, planting is carried out in the forest canopy in the wild Plantation forest is also a solution for developing planting Anoectochilus areas For this planting solution, the selection of planting areas meeting the requirements of natural, climate, soil and socio-economic conditions plays a very important role Cultivation conditions must be similar to the natural conditions of the distribution area In addition to the planting season, it is necessary to avoid periods of bad weather like: Rain, too hot or too cold to avoid risks So with climate conditions like in Hoa Binh, the research team selected the times of cultivation is March, June; September; December The results show that the best planting time is March because March in Hoa Binh has an average temperature of 14-200C and a spring rain, high humidity is suitable for growth and development of A setaceus, furthermore, after 4-6 weeks, the tree is stable until 22 summer when the tree grows the most biomass so that the tree grows well and we can harvest in December, this is also the time for A setaceus flowers the best time for medicinal harvest For crops in June to July and August, when the trees begin to grow, they caught rain and pests cause more damage which results in the lowest survival rate Crops at the time of September have a higher survival rate but the biomass grows slowly due to bad weather conditions and then the cold dry season At the time of December, plants had low survival rates and plants grew slowly due to the weather conditions So the most suitable time for planting Anoectochilus Blume in the North is March - Pests harmful and prevention As we know, the species of Anoectochilus Blume are very sensitive to ecological environment conditions, they are suitable to live in a humid and lowlight condition, which is also an appropriate condition for the proliferation of harmful species When living in natural conditions they are protected by symbiotic and reciprocal relationships So when cultivating Anoectochilus Blume, it is difficult to avoid effects of harmful organisms The studies in Hoa Binh from March 2014 to February 2015 on Anoectochilus show that there are major pest species: Vermilion spider, snail, slug, bacterial soft rot The survey results on the level of harm are shown in Table 3.20 Tabe 3.20: Damage level of major pests and diseases on Anoectochilus Blume (March 2014 - February 2015) Popularity Test period Vermilion spider Snail Slug Bacterial soft rot 3/2013 - + + - 4/2013 + +++ +++ + 5/2013 ++ +++ + ++ 23 6/2013 +++ + - ++ 7/2013 +++ + - + 8/2013 +++ + + + 9/2013 ++ +++ +++ + 10/2013 ++ +++ +++ + 11/2013 ++ +++ ++ + 12/2013 + ++ + - 1/2015 - ++ + - 2/2015 - + ++ + Notes: - No infection; + infection rate ≤ 25%; ++ 25%> infection rate ≥ 50%; +++ 50% ≤ infection rate ≤ 70%; ++++ 70% ≤ infection rate ≤ 100% The above results show that purl is easily attacked by pests and diseases Each pest is adapted to a certain time of year So in order to avoid the losses caused by harmful pests, it is necessary to handle soil carefully and take appropriate measures for each pest The research results show that propagating and culturing solutions are an effective solution to preserve and store Anoectochilus Blume resources in Vietnam 3.4.2 Management and propaganda solutions 3.4.2.1 Management solutions a Bringing the Anoectohilus species to medicinal value as: A annamensis, A setaceus, A elwesii in list of medicinal plants need to be protected in Vietnam b Proposing places with facilities and natural conditions suitable to preserve Anoectochilus Blume species as table 3.21 24 Table 3.21: Proposing sites for conservation of species belonging to the genus Anoectochilus Blume Place Number of species Name of species Sapa Medicinal Station (National Institute of Medicinal Materials Ministry of Health) A setaceus Blume and A calcareus Aver Tam Dao Medicinal Station (National Institute of Medicinal Materials - Ministry of Health) A setaceus Blume; Xuan Lien Nature reserve A annamensis Aver and A elwesii A setaceus Blume and A annamensis Aver Phong Nha Ke Bang National Park Note A setaceus Blume and A annamensis Aver A calcareus Aver Da Lat medicinal center A setaceus Blume and A lylei Rolfe ex Downie 25 Keep in Orchid garden area Keep in Orchid garden area 3.4.2.2 propaganda solutions a Propaganda for managers and investors to see the role and value of Anoectochilus Blume species b Propaganda for the community and management staff at the national parks, nature reserve and localities to help them better understand the value of genetic resources as well as the medicinal value of the Anoectochilus Blume species from which they have a sense of protection and development c Develop a plan for growing area CONCLUSIONS AND RECOMMENDATIONS Conclusions - Collect species name assessment and determine distribution points in which 12 new distribution points of five species are recorded: Anoectochilus annamensis; A calcareus; A elwesii; A lylei and A setaceus of the Orchid family (Orchidaceac) - Extraction and isolation of eight compounds of the A.setaceus species were isolated, of which two compounds were first discovered in A setaceus is adensine and roeoside, a compound is the main flavonoid in the Anoectochilus Blume genus and compounds from the A annamensis, Annamflavone has a strong antimicrobial activity for the first time isolated by nature - The status of exploitation and trade of purl species at national parks has been assessed, nature reserves and other forest showed, households at survey sites are still exploiting and taking part in trading Anoectochilus Blume - Proposing solutions for management of propaganda and technical solutions are propagation and cultivation of three species of A annamensis, A lylei and A setaceus - Successfully propagated by in vitro propagation techniques from seeds and shoots from three species of A annamensis, A.lyle and A setaceus In the in vitro propagation process, liquid medium was used and LED lighting was used as modern technologies to increase propagation efficiency This result can be applied to the development of the Anoectochilus Blume growing area for commercial purposes 26 - Cultivation is carried out by the method of raising in greenhouse after 16 weeks of culture, it grows and developes well (survival rate is over 80%; weight is nearly times) and cultivated under forest canopy in Hoa Binh The most suitable planting time was identified in March, the tree grew and developed after 16 weeks of cultivation (survival rate is over 70%; weight nearly doubled Recommendation It is necessary to soon supplement the Anoectochilus Blume species to the list of valuable medicinal species that need to be preserved and developed Continue to conduct research and survey projects to inventory and supplement the number and quantity of species, the research topics of chemical composition, medicinal value of species belonging to the genus Anoectochilus Blume in Vietnam Researching and experimenting to replicate culturing models in the greenhouse and under the forest canopy for domestic and export markets 27 ... Hoang Su Phi- Ha Giang Bac Me- Ha Giang Tra Linh- Cao Bang Sa Pa- Lao Cai Ky Anh - Nghe An Nam Tra My-Quang Nam A annamensis A calcareus National Park + + + + + + + _ Natural reserve + _ + + Other... 2.1 Research subjects The object of the study is the species of genus (Anoectochilus Blume), distributed in nature in Vietnam Surveying, collecting samples, determining the distribution area and... Morphological characteristics Anoectochilus in VietNam and classification of species on Determination of the active ingredient in two species of loài A setaceus A annamensis Studying and investigating the