GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLGY VU THI HIEN

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLGY ----------------------------- VU THI HIEN REGENERATION AND MICROPROPAGATION OF Panax vietnamensis Ha et Grushv. USING THIN CELL LAYER TECHNOLOGY Major: Plant Physiology Code: 9.42.01.12 SUMMARY OF PHILOSOPHY DOCTORAL DISSERTATION ON BIOLOGY Ho Chi Minh City - 2018

The work was realized in Graduate University of Science and Technology, Vietnam Academy of Science and Technology Advisor 1: Prof. Duong Tan Nhut, Ph.D. Advisor 2: Thai Xuan Du, Ph.D. Reviewer 1:... Reviewer 2:... Reviewer 3:... The thesis will be evaluated by doctoral committee at Graduate University of Science and Technology, Vietnam Academy of Science and Technology on..2018 The thesis is available at: - Library of Graduate University of Science and Technology - National Library of Vietnam

1 INTRODUCTION 1. The necessity of the dissertation Ngoc Linh ginseng is a Vietnamese endemic ginseng with the scientific name Panax vietnamensis Ha et Grushv. Since discovered in 1973, it can be said that Ngoc Linh ginseng is one of the most important medicinal plants. Many reports indicated that Ngoc Linh ginseng has not only the pharmacological characteristics of a Ginseng, but also the individual characteristics such as anti-stress, decrease of depression and anxiety, stimulation of the immune system, resistance to cytotoxic toxins, antioxidant in vitro and in vivo, etc. The success of propagation of Ngoc Linh ginseng is still limited because this species is only grown on Ngoc Linh mountain. To harvest gingseng roots, the propagation period lasts 6 to 7 years to store enough bioactivities. Our thesis entitled "Regeneration and micropropagation of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) using thin cell layer technique" has been carried out. The aim of this study is to obtain a number of vigorous plantlets with and high quality roots and tubers, especially, they are well adapted to the natural conditions, thereby contributing to preserving this precious medicinal plant. 2. Objective The objective of the study was to find the explant resources, the type and concentration of plant growth regulators (), as well as the in vitro culture conditions suitable for different morphogenesis processes - callus induction, direct embryogenesis, shoot and root formation, etc. The growth and development of in vitro Ngoc Linh ginseng derived from thin cell layer (TCL) was examined in Quang Nam to assess the adaptability of in vitro Ngoc Linh plantlets in its natural territory, compared to those growing in Bidoup - Nui Ba National Park, Lam Dong. 3. The contents of the thesis 3.1. Research on the morphogenesis from different explant resources 3.2. Research on growth and subsequent development of plantlets in vitro in different ecological conditions 3.3. Qualitative and quantitative saponin in plant in vitro and at nursery. CHAPTER I OVERVIEW The thesis has consulted 34 Vietnamese documents and 94 English documents and 2 internet documents; (1) Introduction to ginseng; (2) Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.); (3) cell culture technology; (4) factors influencing morphogenesis; (5) plant growth regulators; (6) the role of light in the regeneration, growth and development of plants; (7) The plant regeneration.

2 CHAPTER II RESEARCH OBJECTIVE AND METHODS 2.1. Materials 2.1.1. Plant materials Explant source for morphogenesis: including leaf, petiole and rhizome of 3-month old Ngoc Linh ginseng in vitro plants; Explants grew under different ex vitro conditions: Ngoc Linh ginseng intact plants with rhizomes and leaves, about 3 cm in height; explant sources for determining bioactive agents: in vitro plantlets, 6-month old, 1-year old and 2-years old plants. 2.1.2. Equipment - tools, standard chemicals and solvents Light intensity meter LI-250A Light meter; microscope; Equipment used in HPLC analysis (High Performance Liquid Chromatography, Rg1, Rb1, MR2). Chloroform: methanol: water (65:35:10). 2.2. Research Methods 2.2.1. Plant morphogenesis method 2.2.2. Plant morphology and microscope observation method 2.2.4. Saponin content analysis method 2.2.4.1. Thin layer chromatography method 2.2.4.2. High Performance Liquid Chromatography (HPLC) 2.3. Research establishment methods 2.3.1. Content 1: Researching the morphogenesis from different explant sources 2.3.1.1. Evaluating the effect of single on morphogenesis of leaf explant ttcl_l under light and dark conditions 2.3.1.2. Evaluating the effect of single on morphogenesis of petiole explant ttcl_l under light and dark conditions. 2.3.1.3. Evaluating the effect of single on morphogenesis of ltcl_c petiole explant under light and dark conditions 2.3.1.4. Evaluating the effect of single on morphogenesis of TTCL_R rhizome explant under light and dark conditions 2.3.1.5. Evaluating the effect of the combination of auxin and cytokinin on the morphogenesis of the leaf explant ttcl_l under light and dark conditions. 2.3.1.6. Evaluating the effect of the combination of auxin and cytokinin on the morphogenesis of the ttcl_c petiole explant under light and dark conditions. 2.3.1.7. Evaluating the effect of the combination of auxin and cytokinin on the morphogenesis of the ltcl_c petiole explant under light and dark conditions.

3 2.3.1.8. Evaluating the effect of the combination of auxin and cytokinin on the morphogenesis of the rhizome explant ttcl_r under light and dark conditions. 2.3.1.9. Morphological anatomy 2.3.1.10. Develop Ngoc Linh plantlets from somatic embryos 2.3.2. Research on growth and subsequent development of in vitro plantlets under different ecological conditions 2.3.2.1. Research on the growth and development of Ngoc Linh ginseng cultured in vitro grown in Quang Nam 2.3.2.2. Research on the growth and development of Ngoc Linh ginseng cultured in vitro grown in the Heaven Gate of Bidoup - Nui Ba National Park (Lam Dong) 2.3.3. Content 3: Qualitative and quantitative saponin in ginseng in vitro and in complete ginseng at nursery stage. 2.3.3.1. Determine the content of saponin in Ngoc Linh ginseng in vitro, 6 month ginseng, 1 year and 2 year old seedlings planted in Quang Nam. 2.3.3.2. Quantification of saponins in Ngoc Linh ginseng in vitro, 6 month ginseng, 1 year and 2 year old trees were planted in Quang Nam. 2.4. Statistics The experiment was completely randomized (CDR). The mean of the follow-up indices among the treatment formulas was analyzed by ANOVA method, then compared with the Ducan test at confidence level P <0.05 using SPSS 16.0 software. [58]. 2.5. Culture conditions 2.5.1. In vitro condition 2.5.2. Ex vitro condition 2.6. Location and time of the experiment 3.1. RESULTS CHAPTER III RESULTS AND DISCUSSIONS 3.1.1. Research on the morphogenesis from different explant sources 3.1.1.1. Effect of single on morphogenesis of leaf explant ttcl_l under light and dark conditions The in vitro leaf explants of TTCL_L were inoculated in culture medium. After 10 weeks of culture, results were observed as shown in Table 3.1; 3.2 and 3.1; 3.2.

4 Table 3.1. Effect of single on morphogenesis of leaf explant ttcl_l in photoperiod of 16 hours/day Embryo genesis Callogenes is formation numbe r Cont Necrosis 0 0 e 0 d 0 e 0 e rol TDZ 0.01 0 e 0 d 0 e 0 e Necrosis TDZ 0.05 0 e 0 d 0 e 0 e Necrosis TDZ 0.1 0 e 0 d 0 e 0 e Necrosis TDZ 0.2 0 e 0 d 0 e 0 e Necrosis TDZ 0.5 0 e 0 d 0 e 0 e Necrosis TDZ 1.0 0 e 0 d 0 e 0 e Necrosis BA 0.1 0 e 0 d 0 e 0 e Survive but no induction BA 0.2 0 e 0 d 0 e 0 e Survive but no induction BA 0.5 0 e 0 d 0 e 0 e Survive but no induction BA 1.0 0 e 0 d 0 e 0 e Survive but no induction BA 2.0 0 e 0 d 0 e 0 e Survive but no induction 2,4- D 2,4- D 2,4- D 2,4- D 2,4- D NA A NA A NA A NA A NA A 0.1 0 e 0 d 0 e 0 e 0.2 43.3 d 0 d 0 e 0 e 0.5 70.6 b 44.4 c 0 e 0 e 1.0 86.3 a 97.7 a 42.1 b 2.86 a 2.0 49.6 c 76.6 b 9.9 d 1.06 c 0.1 0 e 0 d 0 e 0 e 0.2 0 e 0 d 0 e 0 e 0.5 0 e 0 d 0 e 0 e 1.0 70.0 b 74.4 b 26.6 c 0.64 d 2.0 89.0 a 97.7 a 63.3 a 2.59 b Necrosis A few global embryos A few global and heart shaped embryos White, brown, and compact callus. Embryos cluster White, brown, and compact callus. Few embryos Necrosis Necrosis Necrosis heart, global shaped embryos. Few short roots. Dark brown callus Heart, global, cotyledon and torpedoe shaped embryos Short white roots, dark brown callus

5 Table 3.2. Effect of single on morphogenesis of leaf explant ttcl_l in dark conditions Embryogenesis formation number Control 0 0 f 0 d 0 d 0 d Necrosis TDZ 0.01 0 f 0 d 0 d 0 d Necrosis TDZ 0.05 0 f 0 d 0 d 0 d Necrosis TDZ 0.1 0 f 0 d 0 d 0 d Necrosis TDZ 0.2 0 f 0 d 0 d 0 d Necrosis TDZ 0.5 0 f 0 d 0 d 0 d Necrosis TDZ 1.0 0 f 0 d 0 d 0 d Necrosis BA 0.1 0 f 0 d 0 d 0 d BA 0.2 0 f 0 d 0 d 0 d BA 0.5 0 f 0 d 0 d 0 d BA 1.0 0 f 0 d 0 d 0 d BA 2.0 0 f 0 d 0 d 0 d 2,4-D 0.1 37.6 e 0 d 0 d 0 d 2,4-D 0.2 84.3 b 39.9 b 0 d 0 d 2,4-D 0.5 71.6 c 96.6 a 73.3 b 2.74 b 2,4-D 1.0 92.0 a 98.8 a 79.9 a 2.87 a Survive. no development Survive. no development Survive. no development Survive, no development Survive, no development Global and heart shaped embryos Global, heart shaped, and cotyledon embryos Global, heart shaped, and cotyledon embryos; white root Global, heart shaped, and cotyledon embryos; white root Yellow and some while 2,4-D 2.0 47.3 d 97.7 a 57.7 c 2.63 c callus; few embryo, white root NAA 0.1 0 f 0 d 0 d 0 d Necrosis NAA 0.2 0 f 0 d 0 d 0 d Necrosis NAA 0.5 0 f 0 d 0 d 0 d Necrosis NAA 1.0 0 f 0 d 0 d 0 d Necrosis NAA 2.0 0 f 13.3 c 0 d 0 d yellow callus at leaf egles

6 3.1.1.2. Effect of single on morphogenesis of petiole explant ttcl_c under light and dark conditions. After 10 weeks culturing, the morphogenicity indicators are presented in Table 3.3 and Figure 3.3. Table 3.3. Effect of single on morphogenesis of petiole explant ttcl_c at photoperiod of 16 hours/day and in dark. Embryogeneis light Callogeneis formation Dark number Contro Necrosis 0 0 e 0 e 0 d 0 d l TDZ 0.01 0 e 0 e 0 d 0 d Necrosis TDZ 0.05 0 e 0 e 0 d 0 d Necrosis TDZ 0.1 0 e 0 e 0 d 0 d Necrosis TDZ 0.2 0 e 0 e 0 d 0 d Necrosis TDZ 0.5 0 e 0 e 0 d 0 d Necrosis TDZ 1.0 0 e 0 e 0 d 0 d Necrosis BA 0.1 0 e 0 e 0 d 0 d Necrosis BA 0.2 0 e 0 e 0 d 0 d Necrosis BA 0.5 0 e 0 e 0 d 0 d Necrosis BA 1.0 0 e 0 e 0 d 0 d Necrosis BA 2.0 0 e 0 e 0 d 0 d Necrosis 2,4-D 0.1 0 e 0 e 0 d 0 d Necrosis 2,4-D 0.2 0 e 0 e 0 d 0 d Necrosis 2,4-D 0.5 0 e 83.3 a 0 d 0 d Soft yellowish callus 2,4-D 1.0 16.6 d 63.3 b 0 d 0 d Soft yellowish callus 2,4-D 2.0 63.3 c 33.3 d 0 d 0 d Brown callus NAA 0.1 0 e 0 e 0 d 0 d Necrosis NAA 0.2 0 e 0 e 0 d 0 d Necrosis NAA 0.5 0 e 0 e 31.3 c 2.0 c White short root NAA 1.0 86.6 a 0 e 75.5 b 6.4 b NAA 2.0 76.6 b 46.6 c 89.9 a 15.5 a Black callus. Some white lateral root Yellowish callus. A lot of white lateral root 3.1.1.3. Effect of single on morphogenesis of ltcl_c petiole explant in light and dark conditions The indicators recorded after 10 weeks of culture are shown in tables 3.4, 3.5 and 3.4, 3.5.

7 Table 3.4. Effect of single on morphogenesis of ltcl_c petiole explant at photoperiod of 16 hours/day. Embryogenesis formation number Control 0 0 e 0 e 0 f 0 c Necrosis TDZ 0.0 Necrosis 0 e 0 e 0 f 0 c 1 TDZ 0.0 Necrosis 0 e 0 e 0 f 0 c 5 TDZ 0.1 0 e 0 e 0 f 0 c Necrosis TDZ 0.2 0 e 0 e 0 f 0 c Necrosis TDZ 0.5 0 e 0 e 0 f 0 c Necrosis TDZ 1.0 0 e 0 e 0 f 0 c Necrosis BA 0.1 0 e 0 e 0 f 0 c Necrosis BA 0.2 0 e 0 e 0 f 0 c Necrosis BA 0.5 0 e 0 e 0 f 0 c Necrosis BA 1.0 0 e 0 e 0 f 0 c Necrosis BA 2.0 0 e 0 e 0 f 0 c Necrosis 2,4-D 0.1 0 e 0 e 0 f 0 c Necrosis 2,4-D 0.2 23 d 24.4 d 25.6 d 0.8 c 2,4-D 0.5 59.6 c 89.9 b 79.9 a 4.7 ab 2,4-D 1.0 86.3 a 97.7 a 85.5 a 6.2 a 2,4-D 2.0 69.6 b 98.8 a 71 b 4.0 b NAA 0.1 0 e 0 e 0 f 0 c Necrosis NAA 0.2 0 e 0 e 0 f 0 c Necrosis NAA 0.5 0 e 0 e 0 f 0 c Necrosis NAA 1.0 58.6 c 84.4 c 10.6 e 3.1 b NAA 2.0 84.0 a 96.6 a 37.7 c 4.0 b Few embryos. Black compact callus Cluster of global, heart shaped and cotyledon embryos Yellow compact callus. Green elongated root Many global, heart, torpedo shaped and cotyledon embryos Brown callus. Yellowish roots Heart and global shaped embryos only at leaf edges Brown compact callus Few transparent white root Large amount of callus. Purple embryo cluster milky white heart and global shaped embryos. Little brown calli

8 Table 3.5. Effect of single on morphogenesis of ltcl_c petiole explant in dark. Embryogenesis formation number Contr Necrosis 0 0 c 0 e 0 g 0 f ol TDZ 0.01 0 c 0 e 0 g 0 f Necrosis TDZ 0.05 0 c 0 e 0 g 0 f Necrosis TDZ 0.1 0 c 0 e 0 g 0 f Necrosis TDZ 0.2 0 c 0 e 0 g 0 f Necrosis TDZ 0.5 0 c 0 e 0 g 0 f Necrosis TDZ 1.0 0 c 0 e 0 g 0 f Necrosis BA 0.1 0 c 0 e 0 g 0 f Necrosis BA 0.2 0 c 0 e 0 g 0 f Necrosis BA 0.5 0 c 0 e 0 g 0 f Necrosis BA 1.0 0 c 0 e 0 g 0 f Necrosis BA 2.0 0 c 0 e 0 g 0 f Necrosis 2,4-D 0.1 0 c 46.6 d 11.0 f 0.34 ef 2,4-D 0.2 0 c 67.7 c 35.5 de 0.91 d 2,4-D 0.5 49.6 b 84.4 b 41.1 cd 0.51 e 2,4-D 1.0 69.6 a 94.4 a 46.6 c 1.87 c Embryogenic calli. Few yellow roots Large amount of yellow and white calli. White root Few global embryos. Brown callus. Few white roots White soft calli. Few embryos. Transparent white root 2,4-D 2.0 0 c 0 e 0 g 0 f Necrosis NAA 0.1 0 c 0 e 0 g 0 f Necrosis NAA 0.2 0 c 0 e 0 g 0 f Necrosis NAA 0.5 0 c 0 e 31.1 e 0.94 d Few white roots Little yellow NAA 1.0 0 c 45.5 d 61.1 b 6.09 b callus. Milky white short roots. Little yellow NAA 2.0 0 c 81 b 94.4a 19.2 a callus Many milky white short roots.

9 3.1.1.4. Effect of the combination of auxin and cytokinin on the morphogenesis of the rhizome explant ttcl_r in light and dark conditions. After 10 weeks, we observe and record the indicators, the results are shown in tables 3.6, 3.7 and Figures 3.6 and 3.7. Table 3.6. Effect of the combination of auxin and cytokinin on the morphogenesis of the rhizome explant ttcl_r at photoperiod of 16 hours/day. Embryogenesis formation number Contr Necrosis 0 0 g 0 f 0 c 0 c ol TDZ 0.01 0 g 0 f 0 c 0 c Necrosis TDZ 0.05 0 g 0 f 0 c 0 c Necrosis TDZ 0.1 0 g 0 f 0 c 0 c Necrosis TDZ 0.2 0 g 0 f 0 c 0 c Necrosis TDZ 0.5 0 g 0 f 0 c 0 c Necrosis TDZ 1.0 0 g 0 f 0 c 0 c Necrosis BA 0.1 0 g 0 f 0 c 0 c Necrosis BA 0.2 0 g 0 f 0 c 0 c Necrosis BA 0.5 0 g 0 f 0 c 0 c Necrosis BA 1.0 0 g 0 f 0 c 0 c Necrosis BA 2.0 0 g 0 f 0 c 0 c Necrosis 2,4-D 0.1 0 g 75.5 c 0 c 0 c 2,4-D 0.2 31.0 f 87.7 b 0 c 0 c 2,4-D 0.5 57.3 d 89.9 b 0 c 0 c 2,4-D 1.0 79.6 b 97.7 a 0 c 0 c 2,4-D 2.0 91.0 a 88.8 b 0 c 0 c NAA 0.1 0 g 0 f 0 c 0 c Necrosis NAA 0.2 71.0 c 0 f 0 c 0 c Little white callus Large amount white callus. Few global shaped embryos. Large amount of brown calli. Cluster of global, and heart shaped embryos Large amount of dark brown calli. Global, heart shaped and cotyledon embryos. Large amount of dark brown calli. Many global, heart shaped and cotyledon embryos. Embryo shaped global, heart, cotyledon, torpedo

10 NAA 0.5 70.6 c 0 f 0 c 0 c NAA 1.0 48.6 e 44.4 e 74.4 b 5.09 b NAA 2.0 47.3 e 67.7 d 83.3 a 9.24 a Global, heart, torpedo shaped and cotyledon embryos. Global and heart shaped embryos. Few white roots Global shaped and cotyledon embryos. Yellowish callus. Short white roots. Table 3.7. Effect of the combination of auxin and cytokinin on the morphogenesis of the rhizome explant ttcl_r in dark. Embryogenesis formation number Control 0 0 d 0 f 0 d 0 c Necrosis TDZ 0.01 0 d 0 f 0 d 0 c Necrosis TDZ 0.05 0 d 0 f 0 d 0 c Necrosis TDZ 0.1 0 d 0 f 0 d 0 c Necrosis TDZ 0.2 0 d 0 f 0 d 0 c Necrosis TDZ 0.5 0 d 0 f 0 d 0 c Necrosis TDZ 1.0 0 d 0 f 0 d 0 c Necrosis BA 0.1 0 d 0 f 0 d 0 c Necrosis BA 0.2 0 d 0 f 0 d 0 c Necrosis BA 0.5 0 d 0 f 0 d 0 c Necrosis BA 1.0 0 d 0 f 0 d 0 c Necrosis BA 2.0 0 d 0 f 0 d 0 c Necrosis 2,4-D 0.1 56.3 b 41.1 c 0 d 0 c 2,4-D 0.2 84.0 a 61 b 24.4 c 0.93 b Global shaped embryos. Yellowish callus. Global, shaped, cotyledon embryos. Yellow callus heart and Few embryo 2,4-D 0.5 46.3 c 91 a 27.7 c 0.69 bc Large amount of yellow calli 2,4-D 1.0 0 d 95.5 a 44.4 b 0.58 bc Brown compact callus Few white roots 2,4-D 2.0 0 d 31 d 0 d 0 c Yellow callus NAA 0.1 0 d 0 f 0 d 0 c NAA 0.2 0 d 0 f Explants turned yellow 28.2 c 1.28 b Short white roots

11 NAA 0.5 0 d 24.4 e 48.8 b 0.59 bc Few roots NAA 1.0 0 d 0 f 52.2 b 1.37 b Few yellow short roots Many white NAA 2.0 0 d 0 f 98.8 a 21.7 a roots and lateral roots 3.1.1.5. Effect of the combination of auxin and cytokinin on the morphogenesis of the leaf explants ttcl_l in light and dark conditions The results obtained after 10 weeks of culture are shown in tables 3.8, 3.9, 3.10, 3.11, 3.12, 3.13; Figures 3.8, 3.9, 3.10. Table 3.8. Effect of the combination of 2,4-D and BA on the morphogenesis of the leaf explants ttcl_l at photoperiod of 16 hours/day. 2,4-D BA 1.0 0.1 100 a White green compact callus 1.0 0.2 100 a Many greenis white callus 1.0 0.5 100 a Little yellow and milky white soft callus 1.0 1.0 93.3 b Little yellowis green compact callus 1.0 2.0 90 c Light yellow soft callus 0.1 1.0 47.7 f Little yellowish brown callus 0.2 1.0 60.0 e Little green and brown compact callus 0.5 1.0 80.0 d Little brown compact callus 2.0 1.0 100 a Large amount of yellow and milky white soft callus Table 3.9. Effect of the combination of 2,4-D and BA on the morphogenesis of the leaf explants ttcl_l in dark 2,4-D BA formation 1.0 0.1 100 a 27.3 a Little yellow and milky white soft callus 1.0 0.2 100 a 25.5 b Large amount of milky white soft callus 1.0 0.5 100 a 0 c Large amount of transparent and milky white soft callus 1.0 1.0 100 a 0 c White and milky white soft callus 1.0 2.0 93.3 b 0 c Brownish yellow soft callus 0.1 1.0 73.3 d 0 c Brownish yellow compact callus

12 0.2 1.0 80.0 c 0 c Little brownish yellow and transparent compact callus 0.5 1.0 93.3 b 0 c White and milky white soft callus 2.0 1.0 93.3 b 0 c White and brownish red soft callus Table 3.10. Effect of the combination of 2,4-D and TDZ on the morphogenesis of the leaf explants ttcl_l at photoperiod of 16 hours/day. 2,4-D TDZ 1.0 0.01 100 a White and yellow soft callus 1.0 0.05 100 a Greenish white and brownish yellow compact callus 1.0 0.1 100 a Large amount of greenish white and redish yellow compact callus 1.0 0.2 100 a Little greenish white and yellow soft callus 1.0 0.5 100 a Little greenish white and redish yellow compact callus 1.0 1.0 100 a White and yellow soft callus 0.1 0.2 80 b Little greenish white and redish yellow compact callus 0.2 0.2 80 b Little green compact callus 0.5 0.2 100 a White and brown soft callus 2.0 0.2 73.3 c Brownish yellow soft callus Table 3.11. Effect of the combination of 2,4-D and TDZ on the explants ttcl_l in dark. of the leaf 2,4-D TDZ 1.0 0.01 100 a Brownish yellow soft callus 1.0 0.05 100 a Little yellow and milky white soft callus 1.0 0.1 100 a Large amount of milky white soft callus 1.0 0.2 100 a Little white and brownish yellow soft callus 1.0 0.5 100 a Transparent white and milky white soft callus 1.0 1.0 86.6 c Little milky white and brownish yellow soft callus 0.1 0.2 80.0 d Little transparent white and brown soft callus 0.2 0.2 93.3 b White and brown soft callus 0.5 0.2 93.3 b Little milky white and brownish yellow soft callus 2.0 0.2 0 e Necrosis

13 Table 3.12. Effect of the combination of NAA and BA on the morphogenesis of the leaf explants ttcl_l at photoperiod of 16 hours/day. NAA BA Morphogenesi s 1.0 0.1 13.3 c Little green compact callus 1.0 0.2 0 d Necrosis 1.0 0.5 0 d Necrosis 1.0 1.0 33.3 b Little brown compact callus 1.0 2.0 0 d Survival but no development 0.1 1.0 0 d Necrosis 0.2 1.0 0 d Necrosis 0.5 1.0 0 d Necrosis 2.0 1.0 60 a Little green compact callus Table 3.13. Effect of the combination of NAA and BA on the morphogenesis of the leaf explants ttcl_l in dark. NAA BA 1.0 0.1 40 e Little transparent white and brownish yellow callus 1.0 0.2 33.3 f Little brown compact callus 1.0 0.5 53.3d Little brown compact callus 1.0 1.0 93.3 b White and brownish yellow soft callus 1.0 2.0 13.3 g Little brownish yellow compact callus 0.1 1.0 0 h Survival but no development 0.2 1.0 0 h Necrosis 0.5 1.0 90.0 c Little greenish white and brownish yellow soft callus 2.0 1.0 100 a White and brown soft callus 3.1.1.6. Effect of the combination of auxin and cytokinin on the morphogenesis of the ttcl_c petiole explant in light and dark conditions. After 10 weeks of culture, the results were observed, recorded and compared the effect of each pair of 2,4-D in combination with BA, 2,4-D in combination with TDZ and BA in combination with NAA. present in tables 3.14, 3.15, 3.16, 3.17, 3.18, 3.19 and Figures 3.11, 3.12, 3.13.

14 Table 3.14. Effect of the combination of 2,4-D and BA on the morphogenesis of the ttcl_c petiole explant at photoperiod of 16 hours/day 2,4-D BA 1.0 0.1 98.8 a Little green and brownish yellow soft callus 1.0 0.2 97.7 a Yellowish white soft callus 1.0 0.5 98.8 a Yellow soft callus 1.0 1.0 97.7 a Milky white, green, and brown compact callus 1.0 2.0 74.4 c Little light yellow compact callus 0.1 1.0 96.6 a Little green compact callus 0.2 1.0 98.8 a Green and white compact callus 0.5 1.0 98.8 a Large amount of green and purple compact callus 2.0 1.0 86.6 b Little yellow soft callus Table 3.15. Effect of the combination of 2,4-D and BA on the morphogenesis of the ttcl_c petiole explant in dark. 2,4-D BA 1.0 0.1 97.7 a Light yellow soft callus 1.0 0.2 98.8 a Large amount of yellow and milky white soft callus 1.0 0.5 97.7 a Milky white and light yellow soft callus 1.0 1.0 97.7 a Little light brown and light yellow soft callus 1.0 2.0 98.8 a Low light brown and light yellow soft callus 0.2 1.0 96.6 a Yellow brown soft callus 0.5 1.0 97.7 a Milky white and light yellow soft callus 2.0 1.0 96.6 a Little yellow soft callus Table 3.16. Effect of the combination of 2,4-D and TDZ on the morphogenesis of the ttcl_c petiole explant at photoperiod of 16 hours/day 2,4-D TDZ 1.0 0.01 100 a Green and white soft callus 1.0 0.05 100 a Dark yellow soft callus 1.0 0.1 100 a Large amount of yellow and milky white soft callus 1.0 0.2 100 a Little yellow and milky white soft callus 1.0 0.5 100 a Little green, brown, and white soft callus

15 1.0 1.0 100 a Little yellow and milky white soft callus 0.1 0.2 100 a Small green compact callus 0.2 0.2 86.6 b Little green, and brownish yellow compact callus 0.5 0.2 100 a Little yellow and milky white soft callus 2.0 0.2 0 c Necrosis Table 3.17. Effect of the combination of 2,4-D and TDZ on the morphogenesis of the ttcl_c petiole explant in dark. 2,4-D TDZ 1.0 0.01 100 a Transparent white and brownish yellow callus 1.0 0.05 100 a Brownish yellow soft callus 1.0 0.1 100 a Large amount of yellow and milky white soft callus 1.0 0.2 100 a Transparent yellow soft callus 1.0 0.5 100 a Brownish redish and yellow soft callus 1.0 1.0 100 a Brownish red soft callus 0.1 0.2 93.3 c Little brown compact callus 0.2 0.2 93.3 c Little yellow soft callus 0.5 0.2 79.9 c White and yellow soft callus 2.0 0.2 100 a Little milky white soft callus Table 3.18. Effect of the combination of NAA and BA on the morphogenesis of the ttcl_c petiole explant at photoperiod of 16 hours/day. NAA BA 1.0 0.1 100 a Necrosis 1.0 0.2 100 a Necrosis 1.0 0.5 100 a Large amount of yellow and white compact callus 1.0 1.0 100 a Yellow and white compact callus 1.0 2.0 100 a Brownish yellow compact callus 0.1 1.0 0 c Necrosis 0.2 1.0 0 c Necrosis 0.5 1.0 0 c Necrosis 2.0 1.0 86.6 b Little brown compact callus

16 Table 3.19. Effect of the combination of NAA and BA on the morphogenesis of the ttcl_c petiole explant in dark. NAA BA 1.0 0.1 100 a Very little brownish yellow soft callus 1.0 0.2 100 a Little yellow and milky white soft callus 1.0 0.5 100 a Large amount dark yellow soft callus 1.0 1.0 100 a Large amount dark yellow soft callus 1.0 2.0 100 a Large amount dark yellow soft callus 0.1 1.0 0 b Necrosis 0.2 1.0 100 a Large amount dark yellow soft callus 0.5 1.0 100 a Large amount dark yellow soft callus 2.0 1.0 100 a Large amount dark yellow soft callus 3.1.1.7. Effect of the combination of auxin and cytokinin on the morphogenesis of the ltcl_c petiole explants in light and dark conditions. The results recorded after 10 weeks of culture shown in tables 3.20 to 3.25 and Figures 3.14, 3.15, 3.16. Table 3.20. Effect of the combination of 2,4-D and BA on the morphogenesis of the ltcl_c petiole explants at photoperiod of 16 hours/day 2,4- D BA Callog enesis Embryogene sis 1.0 0.1 100 a 24.4 c Little brownish red, green and yellow compact callus 1.0 0.2 93.3 b 48.8 a Red and milky white soft callus 1.0 0.5 86.6 c 31.0 b Little white, green and red callus 1.0 1.0 66.7 d 0 d Red, brownish yellow, and light green callus 1.0 2.0 13.3 e 0 d Little light yellow, and red callus 0.1 1.0 86.6 c 0 d Very tittle callus 0.2 1.0 100 a 0 d Little red brown compact callus 0.5 1.0 93.3 b 0 d Green, and yellow callus on cut surface 2.0 1.0 100 a 0 d Little yellow and milky white soft callus on explant surface and two cut surfaces

17 Table 3.21. Effect of the combination of 2.4-D and BA on the morphogenesis of the ltcl_c petiole explants in dark 2,4- D BA Callogen esis Shoot formation 1.0 0.1 100 a 0 b Brownish yellow, and milky white soft callus 1.0 0.2 100 a 21.0 a Large amount of milky white soft callus 1.0 0.5 100 a 0 b Milky white soft callus 1.0 1.0 100 a 0 b Little yellow and milky white soft callus 1.0 2.0 100 a 0 b Light yellow soft callus 0.1 1.0 46.7 d 0 b Little brownish red compact callus 0.2 1.0 80.0 c 0 b 0.5 1.0 93.3 b 0 b Little yellow and brown compact callus on surface and two cut explants Milk white soft callus on explant surface and two cut surfaces 2.0 1.0 100 a 0 b Little yellow and brown soft callus Table 3.22. Effect of the combination of 2.4-D and TDZ on the morphogenesis of the ltcl_c petiole explants at photoperiod of 16 hours/day 2,4-D TDZ 1.0 0.01 86.6 c Little milky white, and transparent white soft callus on one cut surface 1.0 0.05 93.3 b Little milky white soft callus on explant surface and two cut surfaces 1.0 0.1 93.3 b Greenish white compact callus 1.0 0.2 100 a Large amount of milky white soft callus on explant surface and two cut surfaces 1.0 0.5 100 a Greenish white compact callus on one cut surface 1.0 1.0 93.3 b Brownish red and milky white soft callus on two cut surfaces 0.1 0.2 73.3 d Little green compact callus on one cut surface 0.2 0.2 73.3 d Little milky white soft callus on one cut surface 0.5 0.2 93.3 b Large amount of milky white soft callus on one cut surface 2.0 0.2 0 e Necrosis

18 Table 3.23. Effect of the combination of 2.4-D and TDZ on the morphogenesis of the ltcl_c petiole explants in dark 2,4-D TDZ 1.0 0.01 93.3 b Milky white, and brown soft callus on one cut surface 1.0 0.05 100 a Little yellow and milky white soft callus on one cut surface 1.0 0.1 100 a Large amount of transparent and milky white soft callus on two cut surfaces 1.0 0.2 100 a Milky white and brownish yellow soft callus 1.0 0.5 93.3 b Large amount of yellow and milky white soft callus on one cut surface 1.0 1.0 86.6 c Little milky white and yellow soft callus on two cut surfaces 0.1 0.2 0 e Survival but no development 0.2 0.2 80.0 d White and brownish yellow soft callus on explant surface and two cut surfaces 0.5 0.2 100 a White and brownish yellow soft callus on explant surface and two cut surfaces 2.0 0.2 86.6 c Little brownish yellow soft callus on one cut surfaces Table 3.24. Effect of the combination of NAA and BA on the morphogenesis of the ltcl_c petiole explants at photoperiod of 16 hours/day NAA BA 1.0 0.1 60.0 c Little brownish green compact callus on two cut surfaces 1.0 0.2 53.3 d Little brownish green compact callus on one cut surface 1.0 0.5 13.3 f Little brown compact callus 1.0 1.0 46.6 e Little brown compact callus on two cut surfaces 1.0 2.0 80.0 b 0.1 1.0 0 g Necrosis 0.2 1.0 0 g Necrosis 0.5 1.0 0 g Necrosis 2.0 1.0 93.3 a Little green and brown callus on explant surface and two cut surfaces Little white and brown callus on explant surface and one cut surfaces

19 Table 3.25. Effect of the combination of NAA and BA on the morphogenesis of the ltcl_c petiole explants in dark NAA BA Callog enesis Adventitious root formation Number of roots 1.0 0.1 86.6 c 80.0 b 1.1 b 1.0 0.2 100 a 93.3 a 3.9 a 1.0 0.5 93.3 b 7.7 c 0 c Large amount of brownish yellow soft callus on explant surface and one cut surface. Long white root Little yellow and brown soft callus on explant surface and two cut surfaces Long white root Little yellow and white soft callus Long white root 1.0 1.0 86.6 c 0 d 0 c Little yellow soft callus on one cut surface 1.0 2.0 86.6 c 0 d 0 c Brownish yellow soft callus on one cut surface 0.1 1.0 0 d 0 d 0 c Necrosis 0.2 1.0 0 d 0 d 0 c Necrosis 0.5 1.0 0 d 0 d 0 c Survival but no development 2.0 1.0 100 a 0 d 0 c Little milky white soft callus on explant surface and two cut surfaces Long white root 3.1.1.8. Effect of the combination of auxin and cytokinin on the morphogenesis of the ttcl_r rhizome explants in light and dark conditions. After 10 culture weeks, results are shown in Tables 3.26 to 3.31 and Figures 3.17, 3.18, 3.19. Table 3.26. Effect of the combination of 2.4-D and BA on the morphogenesis of the ttcl_r rhizome explants at photoperiod of 16 hours/day 2,4-D BA 1.0 0.1 100 a Large amount of white and brownish yellow compact callus 1.0 0.2 100 a Large amount of white and brownish yellow compact callus 1.0 0.5 100 a Large amount of milky white soft callus

20 1.0 1.0 100 a Large amount of milky white soft callus 1.0 2.0 100 a Little light yellow and greenish white soft callus 0.1 1.0 34.4 e Little brown compact callus 0.2 1.0 66.6 c Little brown compact callus 0.5 1.0 50 d Little brownish yellow soft callus 2.0 1.0 72.2 b Little milky white and brownish yellow compact callus Table 3.27. Effect of the combination of 2.4-D and BA on the morphogenesis of the ttcl_r rhizome explants in dark. Morphogenesi s 2,4-D BA 1.0 0.1 100 a Large amount of white and yellow compact callus 1.0 0.2 100 a Large amount of milky white and yellow compact callus 1.0 0.5 100 a Large amount of milky white friable callus 1.0 1.0 100 a Large amount of milky white friable callus 1.0 2.0 100 a Little milky white and brownish yellow friable callus 0.1 1.0 13,3 e Little yellow compact callus 0.2 1.0 73.3 d Little brown compact callus 0.5 1.0 83.3 c Little milky white and brownish yellow compact callus 2.0 1.0 86.6 b Little milky white and yellow compact callus Table 3.28. Effect of the combination of 2.4-D and TDZ on the morphogenesis of the ttcl_r rhizome explants at photoperiod of 16 hours/day 2,4-D TDZ Shoot formation 1.0 0.01 100 a 0 b White callus 1.0 0.05 100 a 0 b Little white and brownish yellow soft callus 1.0 0.1 100 a 0 b Large amount of greenish white callus 1.0 0.2 100 a 0 b Little yellow and milky white soft callus 1.0 0.5 86.6 b 0 b White and brown soft callus 1.0 1.0 86.6 b 0 b Brownish yellow and greenish white soft callus 0.1 0.2 33.3 d 42 a 0.2 0.2 33.3 d 42 a Some black explants, few greenish white shoots with 3 leaves per shoot Some black explants, few greenish white shoots with 3 leaves per shoot 0.5 0.2 86.7 b 0 b Milky white friable callus with brown explant edges 2.0 0.2 80.0 c 0 b Milky white and yellow compact callus

21 Table 3.29. Effect of the combination of 2.4-D and TDZ on the morphogenesis of the ttcl_r rhizome explants in dark 2,4-D TDZ 1.0 0.01 100 a Large amount of milky white and yellow soft callus 1.0 0.05 100 a Large amount of yellow and milky white soft callus 1.0 0.1 100 a Milky white and yellow soft callus 1.0 0.2 100 a Brown yellow soft callus 1.0 0.5 93.3 b Little white and brownish yellow soft callus 1.0 1.0 80.0 c Little white and brownish yellow soft callus 0.1 0.2 33.3 d Little yellow compact callus 0.2 0.2 80.0 c Little yellow soft callus 0.5 0.2 93.3 b Large amount of yellow and milky white soft callus 2.0 0.2 93.3 b Little milky white soft callus Table 3.30. Effect of the combination of NAA and BA on the morphogenesis of the ttcl_r rhizome explants at photoperiod of 16 hours/day NAA BA 1.0 0.1 100 a Large amount of greenish white callus 1.0 0.2 86.6 b Large amount of yellow, green, and white compact callus at edges of explants 1.0 0.5 86.6 b Little green compact callus 1.0 1.0 80.0 c Little brownish green compact callus 1.0 2.0 50 e Little black and white compact callus 0.1 1.0 0 f Necrosis 0.2 1.0 0 f Necrosis 0.5 1.0 0 f Necrosis 2.0 1.0 73.3 d Little greenish yellow compact callus Table 3.31. Effect of the combination of NAA and BA on the morphogenesis of the ttcl_r rhizome explants in dark NAA BA 1.0 0.1 100 a Little yellow and white soft callus 1.0 0.2 86.7 c Little brown soft callus 1.0 0.5 80.0 d Little white soft callus with brown edges 1.0 1.0 80.0 d Little white, yellow, brown soft callus 1.0 2.0 73.3 e Yellow and brown callus 0.1 1.0 0 g Necrosis 0.2 1.0 0 g Necrosis 0.5 1.0 33.3 f Little dark brown callus 2.0 1.0 93.3 b Yellow and brown compact callus

22 3.1.1.9. Observation of morphological change of Ngoc Linh ginseng somatic embryo The results of anatomy and imaging of cotyledonnic developmental shapes show that the cotyledon obtained from the treatments did not differ in morphology and that it existed in the major forms of spherical, haunted and dicotyledonous. 3.1.1.10. Create a complete and functional plant from somatic embryo After 8 weeks of culture, all plants induced tubers, leaves uniformly developed, morphologically normal and structured similar to those grown in the wild. 3.1.2. Study the growth and development of seedlings in vitro in different ecological conditions 3.1.2.1. The growth and development of Ngoc Linh ginseng cultured in vitro was planted in Quang Nam Table 3.33. Survival, growth and development of Ngoc Linh ginseng in vitro at nursery stage in Ngoc Linh mountain area, Quang Nam province Evaluation time Survival rate Germination rate New rhizome rate 6 months after planting 93 66 20 12 months after planting 91 78 45 18 months after planting 90 87 70 24 months after planting 90 87 100 Note: The percentage of new leaf growth and tuber formation is based on the survival rate of the plant 3.1.2.2. Research on the growth and development of Ngoc Linh ginseng cultured in vitro in the Gate Area of Heaven, Bi Doup Mountain National Park, Lam Dong province Table 3.34. Survival and growth performance of Ngoc Linh ginseng in vitro at the Heaven Gate of Bidoup Mountain National Park - Lam Dong province Evaluation time Survival rate Germination rate New rhizome rate 6 months after planting 70 30 0 12 months after planting 60 40 20 18 months after planting 50 50 30 24 months after planting 50 50 50 Note: The percentage of new leaf growth and tuber formation is based on the survival rate of the plant

23 3.1.3. Quantification of saponins in vitro and ex vitro in Ngoc Linh ginseng 3.1.3.1. Quantification of saponins in vitro in Ngoc Linh ginseng after 6 months, 12 months and 24 months The thin layer chromatographic analysis revealed that samples of Ngoc Linh ginseng were derived from thin cell layer culture (in vitro ginseng, 6-month-old ginseng, 1-year-old and 2-year-old ginseng) There are three saponins Rg1, Rb1, MR2 when the plates run on chloroform: methanol: water = 65:35:10. This proves that in vitro and ex vitro cultures of Ngoc Linh ginseng have full presence of all three types of Rg1, Rb1, MR2. Thus, the qualitative results show that the samples contained saponins. 3.1.3.2. Quantification of saponins in vitro Ngoc Linh ginseng after 6 months, 12 months and 24 months Table 3.35. Saponin content of in vitro and ex vitro Ngoc Linh ginseng Samples Rg 1 Rb 1 MR 2 Total 1 (in vitro plants) 0.68 0.51 0.97 2.17 2 (6-month-old plants) 0.11 0.11 0.47 0.70 3 (1-year-old plants) 0.96 0.85 0.87 2.69 4 (2-year-old plants) 1.02 1.74 0.77 3.54 CONCLUSION AND RECOMMENDATION 1. Conclusion The leaf sample ttcl_l gave the highest embryogenesis (89.6%, 29 cotyledonary emgryos per explant) when explants were cultured on medium supplemented with 2 mg/l NAA under 16-hour light conditions. ltcl_c, ttcl_l were cultured in 1.0 and 2.0 mg/l 2,4-D supplementation in light and dark conditions for the highest callogenesis rate (97,98%). ttcl_r cultured on medium supplemented with 2 mg/l NAA in dark conditions resulted in rooting rate of 98.8% and 21.7 roots / sample. The highest shoot regeneration rate of 42% was found when ttcl_r was cultured on medium supplemented with 0.2 mg/l TDZ in combination with 0.2 mg/l 2,4-D under light conditions. ttcl_l, ttcl_c and ltcl_c, and ttcl_r were cultured on medium supplemented with 2,4-D and BA or TDZ at different concentrations under light conditions and in the darkness gave high callogenesis rate. The embryos obtained in all treatments showed not to be different in they are in global, heart, torpedo shapes and dicotyledons.

24 The cotyledons transferred onto SH medium supplemented with 1 mg/l BA, 0.5 mg/l NAA, 50 g/l sucrose, 9 g/l agar, 1 g/l activated charcoal under illumination at 16h/day, all plants induced rhizomes, well developed leaves, normal leaf morphology and same structure as the plants growing in natural habitats. In addition, 10,000 in vitro plantlets were also collected from the study; Plantlets cultivated in Ngoc Linh Moutain (Quang Nam province) gave the highest survival rate (90%) after 6 months and new leaf formation rate (87%) after 1.5 years compared to plantlets cultivated in Bidoup - Nui Ba National Park, Lam Dong province (70%, 30%; respectively). Especially, 2 year-old plants (cultivated in Quang Nam province) have a microrhizomes (100%) and tends to grow into bushes. The total saponin content was highest when 2-year-old plants grew on Ngoc Linh mountain (3.54%). 2. Recomandation Further investigating effect of the explant types and how to place them on media on the morphogenesis. Further monitoring growth parameters and analyze the saponin content accumulated in Ngoc Linh ginseng derived from thin layer cell culture at different ages. THESIS CONTRIBUTIONS Previous studies on Ngoc Linh ginseng mainly arise based on indirect morphogenesis via callus that extends the culture time and reduces economic efficiency. Application of thin-layer cell culture (TCL) technique to morphogenesis on in vitro explants (leaf, pedicle, and rhizome) of the in vitro Ngoc Linh ginseng will give direct embryogenesis, adventitious shoots and roots, and callus. This will be a new effective method for regenerating Ngoc Linh ginseng in vitro. - Identification of appropriate culture medium for the ability to generate embryos from leaves, shoot regeneration from rhizomes to serve for propagation. - From this sudy, 10,000 Ngoc Linh plantlets were collected from in vitro embryogenesis with an ability of developing well in natural conditions (Ngoc Linh mountain in Quang Nam province and the Heaven Gate in Bidoup - Nui Ba National Park, Lam Dong Province). - Establishment of the protocol in Ngoc Linh ginseng propogation from TCL culture technology and increase the survival of embryos derived from TCL.

25 LIST OF PUBLICATIONS RELATED TO THE THESIS 1. Vu Thi Hien, Vu Quoc Luan, Nguyen Phuc Huy, Nguyen Ba Nam, Bui Van The Vinh, Thai Xuan Du, Duong Tan Nhut. 2014. Direct somatic embryogenesis from leaf, petiole and rhizome explant of Panax vietnamensis Ha et Grushv. Journal of Biology, 36(1SE): 277-282, 2014. 2. Vu Thi Hien, Vu Quoc Luan, Nguyen Phuc Huy, Nguyen Ba Nam, Nguyen Thi Kim Loan, Nguyen Thanh Sang, Vu Thi Thuy, Nguyen Hong Hoang, Thai Xuan Du, Duong Tan Nhut. 2015. Application of Thin Cell Layer Technique in Studying of Panax vietnamensis Ha et Grushv. Journal of Science and Development, 13(4): 661-669, 2015. 3. Vu Thi Hien, Nguyen Phuc Huy, Bui Van The Vinh, Hoang Xuan Chien, Hoàng Thanh Tung, Nguyen Ba Nam, Vu Quoc Luan, Duong Tan Nhut. 2015. Somatic embryogenesis from leaf transverse thin cell layer derived-callus of Vietnamese ginseng (Panax vietnamensis Ha et Grushv.). Journal of Biotechnology, 13(4): 1-11, 2015. 4. Duong Tan Nhut, Hoang Thanh Tung, Vu Thi Hien, Nguyen Ba Nam, Nguyen Phuc Huy, Vu Quoc Luan. 2016. Assessment of the possibility of flowering, fruiting and saponin accumulation of somatic embryoderived Panax vietnamensis Ha et Grushv plants growing in Kon Tum and Quang Nam. Journal of Biotechnology, 14(1A): 263-268. 5. Duong Tan Nhut, Nguyen Phuc Huy, Ngo Thanh Tai, Nguyen Ba Nam, Vu Quoc Luan, Vu Thi Hien, Hoang Thanh Tung, Bui The Vinh, Tran Cong Luan. 2015. Light-emitting diodes and their potential in callus growth, plantlet development and saponin accumulation during somatic embryogenesis of Panax vietnamensis Ha et Grushv. Biotechnology and Biotechnological Equipment, 29(2): 299-308.