THE MINISTRY OF EDUCATION AND TRAINING THE MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAMESE ACADEMY OF FOREST SCIENCES ***** LUU THE TRUNG RESEARCH ON SCIENTIFIC FOUNDATION TO CONSERVE RHODODE[.]
THE MINISTRY OF EDUCATION AND TRAINING THE MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAMESE ACADEMY OF FOREST SCIENCES ***** LUU THE TRUNG RESEARCH ON SCIENTIFIC FOUNDATION TO CONSERVE RHODODENDRON MOULMAINENSE HOOK.f IN LAM DONG SPECIALITY: SILVICUTURAL CODE: 62 02 05 SUMMARY OF FORESTRY PHD DISSERTATION SUPERVISOR: ASSOC PROF PHI HONG HAI ASSOC PROF TRAN VAN TIEN HA NOI: 2022 This work has been completed at the Vietnamese Academy of Forest Sciences Supervisors: Assoc Prof Dr Phi Hong Hai Assoc Prof Dr Tran Van Tien TS Phí Hồng Hải Chairman: Prof Dr Vo Dai Hai Reviewer 1: Assoc Prof Dr Pham Xuan Hoan Reviewer 2: Assoc Prof Dr Tran Minh Hoi Reviewer 3: Assoc Prof Dr Do Huu Son The thesis was defended in front of the institute’s thesis-judging council meeting at the Vietnamese Academy of Forest Sciences at the hour minute day month year 2022 The thesis can be referred at the National Library of Vietnam and the Vietnamese Academy of Forest Sciences PUBLISHED WORKS RELATING TO THE DISSERTATION 1) Luu The Trung, Phi Hong Hai, Tran Van Tien “Characteristics of composition and biodiversity of rhododendron moulmainense Hook f population in Lam Dong”, Forest Science Journal, 2020 (1), p.3-9 2) Luu The Trung, La Anh Duong, Tran Van Tien, Le Ngọc Trieu, Phi Hong Hai “Genetic diversity of naturally distributed rhododendron moulmainense Hook f populations in Lam Vien plateau, Viet Nam revealed by ISSR and SCoT markers”, Malaysian Applied Biology, 2020, 49 (5), pp.41-52 INTRODUCTION The urgency of the dissertation Rhododendron is a genus of beautiful flowering trees that has many values and it is favored by many people In nature, this species are centrally distributed in high area with year-round cool climate There are 80 genera and more than 2,500 species in the genus which are widely distributed in high mountains in tropical, temperate and subtropical Asian countries None is found in dessert and savannas In Vietnam, there are 12 genera and 79 species that mainly live in high mountains (Pham Hoang Ho, 1999); (Vo Van Chi et al, 1978) In Lam Dong in particular, there are around 22 species, centrally distributing in areas at the altitude of more than 1,250m, namely Bidoup - Nui Ba National Park (Do Cao Tri, 2011), Hon Nga under the Protection Forest Management Board of Serepok (Nong Van Duy et al, 2014; Nguyen Thi Thanh Huong et al, 2012a; 2012b) Rhododendron Moulmainense Hook f is a shrub or small tree, 3–7 m tall The leaves are evergreen, elliptical and leathery The flowers are cleistogamous and in umbel The petals are white or pink (Li et al, 2009) This species have many different usage, including roots used in treatment of pulmonary tuberculosis, sputum loss and inflammatories due to falls and anesthetic (Vo Van Chi, 2012; Robert et al, 2018) The plant often grows scattered in the jungle, high mountains in 800 - 2000 m above the sea level, e.g Lai Chau, Lao Cai, Cao bang Da Nang, Kon Tum and Lam Dong (Nguyen Thi Thanh Huong et al, 2011; Vo Van Chi, 2012) However, Rhododendron Moulmainense rather grows in small populations which have quickly declined (more than 50% in the last 10 years) and the decline in the number of individuals in populations continues Accordingly, Rhododendron Moulmainense is listed as vulnerable (VU) A2a; B2b; C2b (iii, iv); D2b (iii, iv) and it is necessary to conserve it for future development (Nong Van Duy et al, 2014) To date, research on Rhododendron Moulmainense are very minimal, limited to describing morphological characteristics, distribution and initial research on breeding However, there is no in-depth, detailed research on distribution, ecological and silvicultural characteristics, breeding and genetic diversity of this species Based on the above-mentioned reasons, the dissertation of: “Research Scientific Foundation to Conserve (Rhododendron moulmainense Hook f.) in Lam Dong” is crucial, bearing scientific and practical values which shall contribute a scientific base for conservation of Rhododendron Moulmainense in Lam Dong Dissertation goals 2.1 General goals Develop some scientific basis to contribute to genetic conservation of Rhododendron Moulmainense in Lam Dong 2.2 Specific goals - Identify characteristics of biological, ecological and genetic characteristics of Rhododendron Moulmainense in Lam Dong; - Identify breeding techniques for Rhododendron Moulmainense for genetic conservation of the species in Lam Dong; - Propose supplementary solutions to conserve Rhododendron Moulmainense in Lam Dong Subjects and scope of the research 3.1 Subject: Rhododendron Moulmainense Hook f 3.2 Scope of the research The research was conducted in forest stands where Rhododendron Moulmainense naturally presents The stands are under the management of Bidoup - Nui Ba National Park, Tuyen Lam Lake tourist and forest management board, and Serepok protection forest management board in Lam Dong province 3.3 Limitations of the dissertation 3.3.1.Limitations of research site In Lam Dong, Rhododendron Moulmainense only distributes in Bidoup, Hon Nga and Tuyen Lam areas with small and uneven sized population in scattered areas Therefore, there are big gaps among permanent sample plots (SPPs) established in distribution areas of the plant 3.3.2.Limitations of breeding research The dissertation only studies propagation by cutting technique 3.3.3.Limitations of research on plantation techniques The dissertation only trials to plant 0.5 to assess the initial growth ability, without fully applying silviculture techniques Scientific and practical values of the dissertation 4.1 Scientific values The dissertation contributes additional information on biological characteristics, distribution, ecological and silvicultural characteristics, genetic diversity, cutting technique and trial plantation of Rhododendron Moulmainense in Lam Dong and in the Central Highlands 4.2 Practical values - The dissertation proposes in-situ and ex-situ conservation measures for Rhododendron Moulmainense in Lam Dong New contributions of the dissertation - Updates on biological characteristics of Rhododendron Moulmainense in Lam Dong - Assessment of biological characteristics of Rhododendron Moulmainense in Lam Dong - Recommendations of breeding and conservation technique through cutting technique for Rhododendron Moulmainense in Lam Dong The dissertation structures The dissertation consists of 129 pages, 28 tables and 30 figures with the following structure: First Part (4 pages); Chapter 1: overview of research problems (25 pages); Chapter 2: content and research methods (23 pages); Chapter 3: research results and discussion (74 pages); The conclusion, existence, and recommendations (3 pages) Chapter OVERVIEW OF RESEARCH PROBLEMS 1.1 WORLDWIDE Rhododendron genus has a scientific name of Rhododendron L., belonging to the rhododendron (Ericaceae) and it was first described by Carl Linnaeus in 1753 in the Genera Plantarum Rhododendron Moulmainense was first published by English botanist Jackson Hooker in 1856 (William et al, 1856) The plant carries a scientific name of Rhododendron Moulmainense Hook.f., originally from Southern China (Fujian, Guangdong, Guangxi, Hong Kong, hunan, Yunnan), Myanmar, Malaysia and Thailand Argent (1998), stated that Rhododendron Moulmainense distributed widely Eastern Asia, from Eastern India to Cambodia, China, and Malaysia, at an altitude of 100-3,000 m 1.2 IN VIET NAM Rhododendron Moulmainense was first recorded by Pham Hoang Ho in 1991 Nguyen Thi Thanh Huong et al (2011) described the plant as up to 15m-high small trees Flowers are 2-3 flowers per axillary bud, white, pink or very light purple, glabrous Fruits are in septicidal capsules, 2.5 to cm long, dark brown Bloom time is between December and April, and fruiting in May to July Following IUCN criteria (2010), Nong Van Duy et al (2014) evaluated Rhododendron Moulmainense as follows: (i) the population has rapidly declined in the last 10 years; (ii) the population distribution is < 100 km and the number of individuals have continuously declined within a population; (iii) the population has become smaller and continuously declined; (iv) fully growth individuals in a population is very few As a consequence, Rhododendron Moulmainense in Lam Dong is classified as VU A2a; B2b; C2b (iii, iv); D2b (iii, iv) Therefore, conservation measures are imperative for future development of the species General discussion: The population of Rhododendron Moulmainense is specified by cluster and narrow distribution Most of recent studies focus on morphologic characteristics and distribution There have been some research on seedling or cutting technique but the numbers of those are very limited However, there is no in-depth, detailed research on distribution, ecological and silvicultural characteristics, breeding and genetic diversity of this species Particularly, wide plantation has not been made Stemming from those practical issues, the dissertation aims to study primarily: - Silvicultural characteristics of Rhododendron Moulmainense, aside from supplementary research on ecological characteristics, population, sideby-side plants, current distribution, plant phenology, soil conditions and plant regeneration in heavily impacted forests in Lam Dong - Diverse genetic populations of Rhododendron Moulmainense in Lam Dong - Propagation of Rhododendron Moulmainense by cutting technique to initiate seedling source for conservation - Some techniques of planting Rhododendron Moulmainense to initially introduce new guidelines for planting and conserving the plant in the local area, making contribution to the restoration and conservation of the species in Lam Dong in particular and in the Central Highland in general Chapter CONTENT AND METHODOLOGY 2.1 RESEARCH CONTENTS 2.1.1 Additional studies in a number of biological and silvicultural characteristics of Rhododendron Moulmainense in Lam Dong • Plant morphology and phenology • Distribution and habitat ecology • Structural features, natural regeneration in the forest stands where the species presents; • Biodiversity characteristics of the forest stands where the species presents 2.1.2 Research on genetic diversity among populations and individuals using theSCoT and ISSR methods 2.1.3 Research on propagation by cutting technique • Effects and concentrations of growth regulators • Effects of seasonality on rooting ability in applying cutting propagation; • Effects of nursery bags' composition and light coverage on survival and growth rate 2.1.4 Initial evaluation of survival and growth rates of Rhododendron moulmainense gene sources in Lam Dong 2.1.5 Proposal of genetic conservation measures for Rhododendron Moulmainense in Lam Dong 2.2 RESEARCH METHOD 2.2.1 Perspective and methods The dissertation was implemented with the main methods below: - Inheritance access, field experiments by area, systematic access, participatory access: 2.2.2 Inheritance data access Selectively access data about natural, socio-economic conditions, forest status maps of three areas where Rhododendron Moulmainense distributes as well as access available conducted research on the species 2.2.3 Supplementary research method to study biological and silvicultural characteristics of Rhododendron Moulmainense 2.2.3.1 Study plant morphology and phenology characteristics Plant morphology: random samples are inspected in areas that Rhododendron Moulmainense naturally grew fully growth and regenerated with D1,3 5% In the remaining sample plots, Rhododendron Moulmainense had IV% index < 5% 3.1.3.2 The relationship between Rhododendron Moulmainense and other species Analysis results shows that Rhododendron Moulmainense has a positive relationship with the Syzygium zeylanicum This is the basis for choosing to grow mixed forest or to enrich the forest, because these species have a mutually supportive relationship when growing and developing 3.1.3.3 Density structure and canopy cover of forest area where Rhododendron Moulmainense grows The density structure of natural forest where Rhododendron Moulmainense grows in the research area ranged from 780-2000 trees/ha The density in Bidoup, Tuyen Lam and Hon Nga ranged from 1,020 to 1,760 trees/ha, 1,000 to 2,000 trees/ha, and 780 to 1040 trees/ha, respectively The canopy closure in the forest stands where Rhododendron Moulmainense grows ranges from 0.5 - 0.9, and 0,68 on average 3.1.3.4 The density structure in the are where Rhododendron Moulmainense grows and exposure direction 13 In research sites, the density of Rhododendron Moulmainense greatly varies from 40 to 260 trees/ha, with the biggest density found in Bidoup Nui Ba ranging from 40 to 260 trees/ha on, and 109 trees on average It followed by the density in Hon Nga, ranging from 40 to 100 trees/ha, and 56 trees on average The least was found in Tuyen Lam with a range of 40 to 60 trees/ha, 50 trees on average Research results show that Bidoup has favorable climate conditions, especially high humidity and advantageous environment, making the density of trees the highest In Tuyen Lam, the humidity is lower and the environment is strongly effected, making considerable decline in the density In addition, the slopiness in Bidoup is lower than that of Hon Nga and Tuyen Lam, which is one of influential factors for distribution density of Rhododendron Moulmainense in Lam Dong These results are rather similar to those studied by Thomson et al (1993), that sloppiness and living conditions are important factors to generate favorable conditions for Rhododendron R Ponticum saplings 3.1.4 Characteristics of natural regeneration where Rhododendron Moulmainense grows 3.1.4.1 Structural assemblage of natural regenerated forest The number of regenerated plants to form the forest structural assemblage ranges from to 12 species Rhododendron Moulmainense is also dominant among regenerated plants in Bidoup However, there are no Rhododendron Moulmainense in Hon Nga 3.1.4.2 Structure of regenerated plants and density of potential regenerated plants Rhododendron Moulmainense often grows in clusters and distributes centrally in small areas Therefore, the results only show assessment of the regeneration density by regions rather than by hectares The regeneration density in the research area ranges from 500 to 2,000 plants/distribution site; the density of potential regeneration starts from 500 plants/site, accounting for 25% to 100% of all regenerated plants in the forest stands In the Bidoup population, the regeneration density is from 500 to 2,000 plants, the potentially regenerated plants accounts for 58.3% of the 14 total in the forest stands, ranging from 25% to 100% In the Tuyen Lam population, the regeneration density is 500 but the potentially regenerated plants highly account for 100% 3.1.4.3 Classification of regenerated plants by quality and origin Most of regenerated plants in research forest stands are in good and average quality Bud regenerated plants has better results than seed regenerated plants In Bidoup, regenerated plants with good quality are from 50 to 100%, and average quality are 25% to 50% Most of the regenerated plants are bud regenerated, accounting for 75% to 100% In Tuyen Lam, regenerated plants with good quality are 100%, and seed regeneration plants are 100% The temporary assessment from research results shows that regenerated plants in research sites are quite good Making a good condition for natural regeneration 3.1.6 Characteristics of biodiversity where Rhododendron Moulmainense grows 3.1.6.1 The abundance and distribution The abundance (A) of populations where Rhododendron Moulmainense grows is significantly different Tuyen Lam population has the highest abundance of 75, following by Bidoup and Hon Nga populations of 69 and 39, respectively Therefore, it proved that living conditions of three naturally grown populations of Rhododendron Moulmainense grows were rather stable, not subject to impacts of human or major changes of environmental conditions The narrow distribution only focus at some places in Lam Dong and currently, those populations are aging without next generation Therefore, it is necessary to have solutions for forest zoning and for promoting regeneration of this species 3.1.6.2 Species biodiversity index (H) and the level of dominance (Cd) The research results show that Shannon's Index (H) in the researched area ranges from 4.672 to 5.254, and 4.89 on average Bidoup population is the highest of 5.254 and Hon Nga the lowest of 4.672 The level of dominance (Cd) ranges from 0.039 to 0.063, and 0.052 on average 3.1.6.3 The Similarity Index or the Sorensen's Index (SI) 15 The Similarity Index among Rhododendron Moulmainense populations significantly vary, e.g Bidoup-Hon Nga is 0,475, Bidoup - Tuyen Lam 0,465 and Hon Nga - Tuyen Lam 0,131 3.2 Genetic diversity of Rhododendron Moulmainense 3.2.1 Genetic diversity of populations and overall species of Rhododendron Moulmainense Genetic diversity of populations and overall species of Rhododendron Moulmainense are determined by 20 ISSR primers, 20 SCoT primers and a combination of ISSR and SCoT The results show that DNA fingerprinting patterns generated with SCoT primers had higher genetic diversity than those generated with ISSR primers, despite the locus generated by the former was much lower than those of the later Specific as follows: 3.2.1.1 Polymorphic locus rates Polymorphic locus rates in Tuyen Lam, Hon Nga and Bidoup within the research are 28.79%, 37.88% and 46.21%, respectively In the general population, the polymorphic locus rate is much higher those of individual populations (PPB = 66.67%) Therefore, polymorphism levels in different populations vary Particularly, Bidoup has the highest polymorphic locus rate, Hon Nga has less and Tuyen Lam has the lowest This is also consistent with the actual survey in the populations The number of species and area of distribution of Rhododendron Moulmainense in Bidoup are higher than those of the two other populations 3.2.1.2 Heterozygosity level The heterozygosity level of Tuyen Lam, Hon Nga and Bidoup and the general populations in the research is 0.0823, 0.1574, 0.2006 and 0.1864 when ScoT is applied; 0.1259, 0.1293, 0.1708 0.163 when ISSR is applied and 0.1041, 0.1434, 0.1857 0.1747 when combining ISSR and SCoT, respectively The result shows that Bidoup has higher heterozygosity than Hon Nga and Tuyen Lam populations, 3.2.1.3 Shannon's Index At the level of populations and species, Shannon's Index obtained at ISSR, SCoT and coordination loci are 0.1220-0.1885 in Tuyen Lam, 0.1947- 16 0.2316 in Hon Ngan 0.2512-0.2907 in Bidoup, and 0.2561-0.2951 in the general population The analysis results enable a statement of highly genetic decline in Tuyen Lam that highly requires special protection measures for this population 3.2.2 Genetic diversity of Rhododendron Moulmainense Analyzing DNA fingerprint patterns generated with ISSR primers shows that the genetic differentiation index (GST) in Rhododendron Moulmainense populations is 0.1290 The number of individuals migrating among populations of a generation is Nm = 3.3774 Analyzing DNA fingerprint patterns generated with SCoT primers shows that the genetic differentiation index (GST) in Rhododendron Moulmainense populations is 0.2124 The number of individuals migrating among populations of a generation is Nm = 1.8539 DNA fingerprint patterns generated by combining both ISSR and SCoT primers show that the (GST) in Rhododendron Moulmainense populations is 0.1375 The number of individuals migrating among populations of a generation is Nm = 2,3824 Thus, the level of genetic differentiation among three Rhododendron Moulmainense populations in Lam Dong is not high 3.2.3 Genetic relations between individuals in populations and the species in overall The genetic similarity coefficient of individuals studied in Tuyen Lam population ranges from 0.841 to 0.985, and 0.937 on average Among those, individual pairs of RhC6 and RhC8, RhC16, RhC17 and RhC18 show the highest genetic differences, while the RhC14-RhC18 and RhC19-RhC20 show the highest genetic similarities The genetic similarity coefficient of individuals studied in Hon Nga population ranges from 0.727 to 1.000, and 0.911 on average The RhB4-RhB18 shows the greatest genetic differences and the RhB12-RhB13 shows the greatest genetic similarities The genetic similarity coefficient of individuals studied in Bidoup population ranges from 0.705 to 0.985, and 0.889, on average, which is lower than those of Tuyen Lam and Hon Nga populations The RhA6-RhA4, and RhA6-RhA5 17 show the greatest genetic differences and the RhA1- RhA2 and RhA4-RhA5 show the greatest genetic similarities 3.3 Study cutting propagation techniques of Rhododendron Moulmainense 3.3.1 Effects of powder and its concentration on rooting rates, length of roots, numbers of root/cutting of Rhododendron Moulmainense The experiment was conducted within six moths Some cuttings in different formulas had roots after 90 days The experimental results are presented in Table 3.19 Table 3.19: Effects of powder and its concentration on rooting rates, length of roots, numbers of root/cutting of Rhododendron Moulmainense Experimental Formula IAA 0.5% IAA 1.0% IAA 1.5% IAA 200% IBA 0.5% IBA 1.0% IBA 1.5% IBA 2.0% NAA 0.5% NAA 1.0% NAA 1.5% NAA 2.0% Control 0% P-value Numbers of experimental cuttings 90 90 90 90 90 90 90 90 90 90 90 90 90 Rooting rate (%) Root length average (cm) Average roots/cutting 6.7fgh 13.3cde 15.6bcd 15.6bcd 13.3cde 24.4a 10.0efg 16.7bc 15.6bcd 18.9b 11.1def 5.6gh 2.2h 0.0000 0.2 0.4 0.2 0.1 0.2 0.2 0.6 0.1 0.4 0.2 0.1 0.2 0.1 0.0000 8.5 7.7 9.0 4.2 7.3 7.2 8.7 4.4 9.7 6.3 5.3 9.0 1.0 0.0001 The results show that the survival rate is quite low, with an average of 7.8-34.4% Rooting rates ranging from 2.2%to 24.4% The highest is IBA 1% reaching 24.4% following by NAA 1% reaching 18.9% and the lowest is the controlling fomular of 2.2% 3.3.2 Effects of liquid substances and its concentration on rooting rates, length of roots, numbers of root/cutting of Rhododendron Moulmainense ... different soil composition, e.g Formula (50% soil + 50% coconut fibers), formula (25% soil + 75% coconut fibers), formula (100% coconut fibers), and formular (100% soil) 30 scion used in each formula... names of species, total height (Hvn) from (0.5m < Hvn ≤ 2m) Quality of regenerated plants are set into levels: Good (G), Average (A) and Poor (P) • Identify potential regenerated plants (Ntstv) (plant/ha)... Department of Biology, University of Dalat for DNA analysis through a) DNA extraction b) DNA analysis c) DNA amplification d) DNA fingerprint records with ISSR and SCoT primers e) Data analysis