Luận án nghiên cứu cơ sở khoa học bảo tồn nguồn gen các loài thuộc chi lan kim tuyến (anoectochilus blume) tại tỉnh thanh hoátt

1 MINISTRY OF MINISTRY OF EDUCATION AND AGRICULTURE AND RURAL TRAINING DEVELOPMENT VIETNAM NATIONAL UNIVERSITY OF FORESTRY NGUYEN TRONG QUYEN RESEARCH ON SCIENTIFIC BASIS OF GENETIC RESOURCES CONSERVATION OF Anoectochilus Blume SPECIES IN THANH HOA PROVINCE Major: Forest resource management Code: 9620211 SUMMARY OF PHD DISSERTATION HANOI, 2022 Research work is completed at: Vietnam National University of Forestry Trường Đại học Lâm nghiệp Việt Nam Scientific supervisors: Scientific supervisor 1: Prof Dr HOANG VAN SAM Scientific supervisor 2: Assoc Prof Dr BUI VAN THANG Chương Reviewer 1: Reviewer 2: Reviewer 3: The defense will be taken in front of the Institutional Board of Dissertation Evaluation at: Vietnam National University of Forestry At: time, Date January Year 2022T hn The dissertation can be found in the libraries: National Library; Vietnam National University of Forestry Library INTRODUCTION Rationale of the study The genus Anoectochilus Blume belongs to the family Orchidaceae, is a valuable plant genus for medical treatment of many diseases, for examples chest pain, abdominal pain, diabetes, nephritis, fever, high blood pressure, paralysis positive, liver disorders… (Nguyen Tien Ban, 2005) [6] Traditional Chinese medicine used these plants to treat diabetes, dissolve tumors, reduce blood lipase and cure hepatitis (Do Tat Loi, 2004) [27] In decree 06/2019/ND-CP, species of this genus are classified in group IA (A setaceus Blume, A.acalcaratus Aver, A.calcareus Aver) and the other species are in group IIA[12] The genus Anoectochilus Blume is facing the risk of habitat loss, degradation, being used as food by animals, and indiscriminate exploitation leading to a decline in natural genetic resources Zhang et al., 2013) [102] In Thanh Hoa, many forest ranges in some National Parks and Nature Reserves could not control the illegal exploitation of Anoectochilus species and they not know exactly how many Anoectochilus species in the area under their management (later confirmed by Phan Xuan Binh Minh, 2019)[28] Due to those reasons, the topic “Research on scientific basis of genetic resources conservation of Anoectochilus Blume species in Thanh Hoa province” was choosen Objectives of the study Providing a scientific foundamental for conservation and development of Anoectochilus Blume species (Orchidaceae) in Vietnam in general and in Thanh Hoa in particular; Chapter LITERATURE REVIEW 1.1 The species diversity of Anoectochilus Blume China is the country with the earliest and most research on the genus Anoectochilus According to the Chinese Botanical Book (2009), [58] there are about 30 species in the genus Anoectochilus Blume Chuan Gao (2009) [56] mentioned that the genus Anoectochilus Blume has about 40 species Summary of species of the genus Anoectochilus Blume from the website of the Botanical List [65], there are 50 species in the world In Vietnam, according to Pham Hoang Ho (2000) [19], there are species in the genus Anoectochilus Blume Nguyen Thien Tich (2001) [40] concluded that there are 10 species in Vietnam Leonid V Averyanov and L Averyanov (2003) [1] mentioned that Vietnam has 13 species Nguyen Tien Ban (2005) [6], Vietnam has 12 species L.Averyanov et al (2008) [48], again announced that there are only 07 species in Vietnam 1.2 Research on genetic resources conservation of Anoectochilus Blume species 1.3 Research on medicinal value and medicinal properties of species of the genus Anoectochilus Blume 1.4.1 Techniques for propagation 1.4.2 Techniques for planting of Anoectochilus formosanus Hayata 1.4.3 Genetic research 1.5 Overview of conservation and information on special-use forests with species distribution of Anoectochilus Blume in Thanh Hoa province Chapter CONTENTS, LOCATION AND METHODS OF STUDY 2.1 Study contents - The diversity of species composition of the genus Anoectochilus Blume in Thanh Hoa - Systematize information on morphology, ecology, distribution, conservation value, key table to identify species of the genus Anoectochilus Blume in Thanh Hoa - Genetic characteristics of A.formosanus - In vitro propagation techniques, planting A.formosanus under the forest canopy and planting in net houses; Analysis and evaluation of total flavonoid content - Proposing solutions for conservation of species of the genus Anoectochilus Blume in Thanh Hoa 2.2 Location of the study In 11 special-use forests in Thanh Hoa province, Vietnam National University of Forestry, Department of Analytical Chemistry, Institute of Medicinal Materials 2.3 Study methods 2.3.1 Method of inheriting documents 2.3.2 Professional solution 2.3.3 Interview method 2.3.4 Field investigation and data collection methods 2.3.5 Distribution map construction method 2.3.6 Genetic characteristics of A.formosanus - Method of extraction of total DNA, Method of analyzing genetic diversity by RAPD technique - Nucleotide sequencing method - Methods of analyzing barcoded DNA data 2.3.7 Chemical analytical methods 2.3.7.1 Machines and equipments 2.3.7.2 Solvents and chemicals 2.3.7.3 Implementation process a) Thin layer chromatography for qualitative analysis b) Quantification of total flavonoids by UV-Vis spectrophotometric method 2.3.8 Experimental method 2.3.8.1 In vitro propagation * Determine the sample sterilization formula * Rapid multiplication of shoots * Planting on different substrates 2.3.8.2 Planting techniques of Anoectochilus formosanus Hayata a) Planting techniques in net houses - Seedlings: Propagated by tissue culture method b) Planting techniques under the forest canopy - Seedlings: Propagated by tissue culture method 2.3.8.3 Methods of measuring growth, monitoring pests and diseases in experiments 2.3.9 Statistical analyses Statistical analyses were carried out using the software SPSS (version 20.0) and Excel Chapter RESULTS AND DISCUSSIONS 3.1 The diversity of species composition of the genus Anoectochilus Blume in Thanh Hoa 3.1.1 Results of the investigation and assessment of species diversity The research results showed that 05 species are distributed in Thanh Hoa province, including: Anoectochilus calcareus Aver), Anoectochilus elwesii (C.B.Clarke ex Hook.f.) King & Pantl), Anoectochilus setaceus Blume, Anoectochilus annamensis Aver and Anoectochilus formosanus Hayata In which, the species of Anoectochilus formosanus Hayata is added to the flora of Vietnam Table 3.1 Coordinates with recorded species of the genus Anoectochilus Blume in Thanh Hoa Research areas Species Pu Hu Nature Reserve A setaceus Blume Pu Luong Nature Reserve A calcareus Aver A elwesii King & Pantl A setaceus Blume Xuan Lien Nature Reserve A.annamens is Aver A setaceus Blume Results Rege Coordinate nera (VN2000) ted tree 488.612/ 2.268.132 488.361/ 2.265.715 510.755/ 2.269.585 Matur e tree Total numbe r of trees 960 IIIa2 2 995 IIIa2 650 IIIb 2 800 IIIb 1385 IIIb 1386 IIIb 2 300 G-N 1 300 G-N 523.978/ 2.260.621 508.497/ 2.265.422 498.916/ 2.208.824 520.912/ 2.199.581 520.870/ 2.199.629 Mean elevatio n (m) Forest state Nam Dong rare and precious gymnospe rms conservati on area A calcareus Aver A.elwesii King & Pantl A setaceus Blume A calcareus Aver A.formosan us Hayata 498.508/ 2.208.381 2 1400 IIIb 488.264/ 2.246.352 3 980 IIIb 1 1065 IIIb 1100 IIIab 1200 IIIb 5 1215 IIIb, N- 488.883/ 2.246.342 488.370/ 2.245.893 487.695/ 2.245.861 487.521/ 2.247.615 IIIb A annamensis Aver A formosanus Hayata Ben En National Park Cuc Phuong National Park (in Thanh Hoa ony) Xuan Lien Nature Reserve Pu Luong Nature Reserve Pu Hu Nature Reserve Tam Quy Nature Reserve Nam Dong rare and precious gymnosperms conservation area Ham Rong cultural and historical relic site Lam Kinh cultural and historical relic site Truong Le cultural and historical relic 10 site 11 Ba Trieu historical relic site A setaceus Blume Special-use forests A elwesii King & Pantl No A.calcareus Aver 3.1.2 Anoectochilus Blume species distribution Table 3.1 Anoectochilus Blume species distribution Anoectochilus Blume in Thanh Hoa N N N N N N N N N N Y Y N N N Y N N Y Y Y N Y N N N N N N N Y Y Y N Y N N N N N N N N N N N N N N N N N N N N * Note: Y natural distribution; N unnatural distribution 3.2 Species morphology characteristics 3.2.1 Anoectochilus annamensis Aver Figure 3.1 A.annamensis Aver was collected and cultivated in the study area of Van Xuan commune, Thanh Hoa province Figure 3.2 Natural distribution of Anoectochilus annamensis Aver in Thanh Hoa province 3.2.2 Anoectochilus calcareus Aver Figure 3.3 A.calcareus gene source in Thanh Hoa Figure 3.4 Natural distribution of the species A.calcareus 10 3.2.3 Anoectochilus elwesii (C.B.Clarke ex Hook.f.) King & Pantl Figure 3.5 A elwesii gene source in Thanh Hoa Figure 3.6 Natural distribution of the species A.elwesii) in Thanh Hoa 3.2.4 Anoectochilus formosanus Hayata * Morphology characteristics: Figure 3.7 Anoectochilus formosanus Hayata) natural gene source in Nam Dong rare and precious gymnosperms conservation area Figure 3.8 Distribution of the species A.formosanus Hayata 13 11 12 13 14 OPB18 OPD11 RA46 RA142 Tổng TB 0,44 0,30 0,45 0,21 0,27 10 80 53 66,7 80,0 85,7 66,7 66,3 3.3.1.2 Analysis of genetic relationships among 10 samples of A.formosanus Table 3.5 Genetic similarity coefficient when compared by pair of 10 samples of A.formosanus KT1 KT2 KT3 KT4 KT5 KT6 KT7 KT8 KT9 KT10 KT1 0.654 0.765 0.802 0.691 0.790 0.765 0.716 0.654 0.753 KT2 KT3 KT4 KT5 KT6 KT7 KT8 KT9 KT10 1.000 0.765 0.753 0.716 0.765 0.765 0.741 0.753 0.753 1.000 0.790 0.802 0.802 0.753 0.802 0.840 0.815 1.000 0.691 0.864 0.864 0.765 0.704 0.901 1.000 0.704 0.654 0.704 0.864 0.691 1.000 0.827 0.827 0.741 0.914 1.000 0.753 0.667 0.840 1.000 0.691 0.790 1.000 0.753 Figure 3.11 Tree diagram showing the genetic relationships of 10 samples of A.A.formosanus 3.3.2 Analysis of some barcoded DNA sequences of A.formosanus 3.3.2.1 Results of cloning gene matK, rbcL and ITS2 sequences 14 M M M a b c a) Nucleotide sequence of the matK gene 3.3.2.2 Sequence analysis of barcoded DNA fragments Figure 3.12 Phylogenetic tree between species A.formosanus and others based on the comparison of nucleotide sequences of the matK gene published on the NCBI International Gene Bank Table 3.6 Abnormal nucleotide positions in the matK gene sequence of A.formosanus with gene codes published in the NCBI International Gene Bank Abnormal nucleotide positions Speciesi A formosanus Hayata nghiên cứu A formosanus A roxburghii A koshunensis A albolineatus A lylei A elatus L discolor ID in NCBI MW6786 27 EU79751 3.1 KY96670 8.1 EU79751 2.1 JN166018 JN166019 KU68709 8.1 AJ543911 1 2 8 6 3 4 8 6 7 7 A T T G C A T A T T C G A C T T G G A T T G C A T A T T T G A C T T G G A T T G C A T A T T C A A C T T A G A T T G C A T _ T T C A A C T T A G T T T T C A T A G T C A G C T T G T T T T G C A T A G T C A G C T T T G T T T T C A T A G T C A G C T T G T T G C G T G C A G G C A A A C A G G 15 b) Nucleotide sequence of the rbcL gene Figure 3.13 Phylogenetic tree between species A.formosanus and other orchid species based on comparison of rbcL gene nucleotide sequences published on NCBI International Gene Bank Table 3.7 Abnormal nucleotide positions in the rbcL gene sequence of A.formosanus with gene codes published in the NCBI International Gene Bank Species A.formosanus A emeiensis A elatus A qeniculatus A lanceolatus L discolor ID in NCBI MW678628 LC057212.1 KU687104.1 JN166036.1 MK451850.1 MH749095.1 c) Nucleotide sequence of the ITS2 gene Abnormal nucleotide positions 161 G G G G A A 383 A A C C C C 16 Figure 3.14 Phylogenetic tree between species A.formosanus and other orchid species based on comparison of ITS2 gene nucleotide sequences published on NCBI International Gene Bank Table 3.8 Abnormal nucleotide positions in the ITS2 gene sequence of A.formosanus with gene codes published in the NCBI International Gene Bank No Species ID A.formosanus nghiên cứu A.formosanus A albolineatus A lylei A.koshunensis Abnormal nucleotide positions 10 112 221 231 273 MW663932 - G C T G GQ396668.1 JN166058.1 JN166060.1 EU700340.1 A A A A T T T T C T C C T A T T G G G A 3.4 In vitro propagation techniques Table 3.9 Sampling location of A.formosanus in Nam Dong rare and precious gymnosperms conservation area (Thanh Hoa) Reasearch areas Date 15/7/2017 16/7/2017 17/7/2017 Areas Nam Dong Nam Dong Nam Dong Results Regenerated tree Mature tree Total number of trees 32 37 486.898/2.246.200 46 52 486.753/2.246.324 53 61 Coordinate (VN2000) 487.229/2.246.024 3.4.1 Sample sterilization and shoot regeneration Table 3.10 Effect of sterilization time on clean explants and shoot regeneration of A.formosanus Fomular KT1 KT2 KT3 KT4 Sig Time (minute) Number of Clean sample clean samples percentages regenerated (%) buds 0 13,33 46,67 14 73,33 0,0001 Percentage of clean samples regenerated buds (%) 13,33 46,67 10,00 0,0001 17 3.4.2 Fast multiplication bud * Effect of mineral environment on shoot multiplication: Table 3.11 Effect of mineral environment on shoot multiplication of A.formosanus Culture medium Percentage of Average Number of multi-bud number of buds promising samples (%) /sample buds Average height of promising buds (cm) 1/2MS 80,00 3,10 ± 0,19b 2,15 ± 0,08b 2,17 ± 0,07a MS Knud* Sig 93,33 66,67 0,036 5,31 ± 0,16a 2,33 ± 0,14c 0,0001 3,54 ± 0,12a 1,31 ± 0,12c 0,0001 1,88 ± 0,08ab 1,54 ± 0,14b 0,001 1/2MS MS Knud Figure 3.15 A.formosanus shoot clusters on 1/2MS, MS and Knud culture media after weeks * Effect of phytohormones shoot multiplication: Table 3.12 Effect of combinations of phytohormones on shoot multiplication of A.formosanus Phytohormone (mg/l) Number of buds/sample Percentage of samples Average Average Formula producing number of BAP Kinetin NAA multiple number of buds/ promising sample shoots (%) buds/sample ĐC SM1 SM2 SM3 SM4 Sig 0,5 0,5 0,7 0,7 0,3 0,3 0,2 0,2 0,3 0,1 0,3 0,1 43,33 100 100 100 100 1,47 ± 0,1e 4,69 ± 0,17d 5,610 ± 0,14c 6,27 ± 0,17b 8,75 ± 0,14a 0,0001 1,29 ± 0,09d 2,15 ± 0,07c 3,28 ± 0,08b 3,51 ± 0,07b 5,04 ± 0,07a 0,0001 Average height of promising buds (cm) 3,05 ± 0,27a 2,18 ± 0,09b 2,30 ± 0,13b 2,39 ± 0,13b 2,35 ± 0,11b 0,022 18 a b d c Figure 3.16 A.formosanus buds on different media after weeks of culture (a- SM1, b-SM2, c-SM3, d- SM4) * Results of creating complete plants from shoots in vitro: Table 3.13 Rooting ability of A.formosanus buds in vitro Formula R0 R1 R2 R3 R4 R5 R6 Sig Culture medium NAA (mg/l) MS 1/2MS 1/2MS 1/2MS MS MS MS 0,5 1,0 1,5 0,5 1,0 1,5 a Percentage Average of buds number of taking root roots /budss (%) 100 1,24 ± 0,12e 100 2,19 ± 0,08d 100 2,49 ± 0,10bcd 100 2,61 ± 0,14bc 100 2,33 ± 0,13cd 100 2,83 ± 0,08b 100 3,64 ± 0,08a 0,0001 Average root length (cm) 0,76 ± 0,06e 1,19 ± 0,04d 1,34 ± 0,07cd 1,68 ± 0,07b 1,32 ± 0,07cd 1,56 ± 0,10bc 2,07 ± 0,12a 0,0001 b Figure 3.17 Completed A.fomorsanus plants on different media: a- R3 (6 weeks), b- R6 (6 weeks) 3.4.3 Effect of type of media on the growth of tissue 19 Table 3.14: Effects of different types of subtrates on the viability and growth of A.fomorsanus after weeks of crowning Substrate Alive Growth height Diameter (mm) ratio (%) (cm) Tree characteristics 66,7% 4,634 ± 0,03c 1,748 ± 0,0016b Thin tree, light green leaves, Coir 83,3% 4,838 ± 0,06b 1,869 ± 0,0014b Thick tree, green leaves, big leaves Dried moss 96,7% 4,9203 ± 0,04a 1,923 ± 0,0026a Thick, tall tree, big dark green leaves Golden sand small Sig 0,01 0,001 a 0,005 b c Figure 3.18 A.formosanus tissue tree grown on different substrates after weeks of crowning (a- Golden sand, b-Coconut coir, c- Dried moss) 3.5 Planting techniques of Anoectochilus 3.5.1 Planting techniques of A.formosanus in net houses 3.5.1.1 Effect of substrate on growth of A.formosanus in net house Table 3.15: Effect of type of substrate on growth of A.formosanus after 12 weeks of growing in net houses Alive CTTN Dried moss Coir Mix Ferns Golden sand Sig percentage (%) 93,3 Height (cm) Diameter (mm) 90 76,7 56,7 66,7 5,3042±0,2390a 5,1028±0,1375ab 4,8628±0,2203ab 4,7246±0,2261b 4,3016±0,0159b 1,8031±0,0087a 1,7558±0,0384ab 1,7168±0,0266bc 1,6844±0,0031bc 1,6049±0,0366c 0,041 0,031 0,004 20 a b d c e Figure 3.19 A.formosanus on different substrates a- Golden sand, b-Coir, c-Dried moss, d- Ferns, e- Mix 3.5.1.2 Effect of planting density on the growth of A formosanus Table 3.16: Effect of density on growth of A.formosanus CTTN MĐ (5x5cm) MĐ (10x10cm) MĐ (15x15cm) Sig Alive percentage (%) 90,0 83,3 86,7 Height (cm) Diameter (mm) 5,2610±0,2170 5,0459±0,2834 4,8836±0,1814 0,5458 1,9616±0,0368 1,8408±0,0353 1,8175±0,0797 0,2152 3.5.1.3 Effects of some foliar fertilizers on the growth and development of A formosanus Table 3.17 Effect of fertilizers on the growth of A.formosanus CTTN VINASUBOR fertilizer Grow More fertilizer HVP(NPK30-10-10) fertilizer N-P-K fertilizer Sample Sig Alive Diameter (mm) percentage Height (cm) (%) 96,7 5,9207±0,0366a 1,9500±0,0111a 83,3 5,2763±0,0722b 1,8997±0,0415a 80,0 5,2677±0,2596b 1,8363±0,0326ab 80,0 76,7 5,2497±0,1083b 1,7680±0,0531b 4,6120±0,2784c 1,7653±0,0369b 0.007 0.022 From the above results, it shows that A fomorsanus is suitable for bio-organic fertilizer application 3.5.1.4 Effect of season on growth of A.formosanus in net houses 21 Table 3.18 Effect of season on growth of A.formosanus after 12 weeks of growing in net houses Season Spring Summer Autumn Winter Sig Alive percentage (%) 90,0 73,3 90,0 80,0 0,217 Height (cm) Diameter (mm) 5,7320±0,2007a 5,1903±0,0381a 5,2194±0,0326ab 5,4392±0,0295b 0,021 1,8826±0,0301a 1,9069±0,0084a 1,8056±0,0036b 1,7667±0,0088b 0,001 3.5.1.5 Results of growing A formosanus in net houses Table 3.19 Growth of A.formosanus after 12 months of growing in net houses Month 1st month 2nd month 3rd month 4th month 5th month 6th month 7th month 8th month 9th month 10th month 11st month 12nd month Sig a Alive percentage (%) 93,3 90,0 87,7 87,7 86,7 86,7 86,7 83,3 83,3 83,3 83,3 83,3 0,999 Height (cm) Diameter (mm) 4,320±0,2627a 4,980±0,2879ab 5,467±0,2973b 5,693±0,3002bc 6,360±0,2551cd 6,743±0,1622de 7,067±0,2381de 7,490±0,2082e 8,393±0,2019f 9,160±0,2212g 11,023±0,2660h 12,907±0,3154i 3,07E-17 1,680±0,0603a 1,743±0,0517ab 1,830±0,0503ab 1,890±0,0651bc 1,990±0,0603cd 2,083±0,0467de 2,173±0,0371ef 2,243±0,0722fg 2,373±0,0384g 2,540±0,0458h 2,683±0,0384i 2,827±0,0376k 4,80E-14 b c Average fresh weight (g/plant) 0,78 g 3,07 g/plant d Figure 3.20 A.formosanus in net house (a: seedlings ;b,c, d: 12 month age plants) 22 3.5.1.6 Pests and diseases of A.formosanus grown in net house Table 3.20 Harmful insect components of A.formosanus Component Thrips Snails Grasshopper ID + + Frequency Very few (