Regeneration and micropropagation of panax Vietnamensis ha et grushv. using thin cell layer technology

MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING 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 (PGRs), 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 PGRs on morphogenesis of leaf explant tTCL_L under light and dark conditions 2.3.1.2. Evaluating the effect of single PGRs on morphogenesis of petiole explant tTCL_L under light and dark conditions. 2.3.1.3. Evaluating the effect of single PGRs on morphogenesis of lTCL_C petiole explant under light and dark conditions 2.3.1.4. Evaluating the effect of single PGRs 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 CHAPTER III RESULTS AND DISCUSSIONS 3.1. RESULTS 3.1.1. Research on the morphogenesis from different explant sources 3.1.1.1. Effect of single PGRs 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 PGRs on morphogenesis of leaf explant tTCL_L in photoperiod of 16 hours/day PGRs (mg/l) Embryo genesis (%) Callogenes is (%) Root formation (%) Root numbe r Remarks Cont rol 0 0 e 0 d 0 e 0 e Necrosis 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 0.1 0 e 0 d 0 e 0 e Necrosis 2,4- D 0.2 43.3 d 0 d 0 e 0 e A few global embryos 2,4- D 0.5 70.6 b 44.4 c 0 e 0 e A few global and heart shaped embryos 2,4- D 1.0 86.3 a 97.7 a 42.1 b 2.86 a White, brown, and compact callus. Embryos cluster 2,4- D 2.0 49.6 c 76.6 b 9.9 d 1.06 c White, brown, and compact callus. Few embryos NA A 0.1 0 e 0 d 0 e 0 e Necrosis NA A 0.2 0 e 0 d 0 e 0 e Necrosis NA A 0.5 0 e 0 d 0 e 0 e Necrosis NA A 1.0 70.0 b 74.4 b 26.6 c 0.64 d heart, global shaped embryos. Few short roots. Dark brown callus NA A 2.0 89.0 a 97.7 a 63.3 a 2.59 b Heart, global, cotyledon and torpedoe shaped embryos Short white roots, dark brown callus * Different letters within a column indicate significant differences at P<0.05 by Duncan’s multiple range tests. 5 Table 3.2. Effect of single PGRs on morphogenesis of leaf explant tTCL_L in dark conditions PGRs (mg/l) Embryogenesis (%) Callogenesis (%) Root formation (%) Root number Remarks 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 Survive. no development BA 0.2 0 f 0 d 0 d 0 d Survive. no development BA 0.5 0 f 0 d 0 d 0 d Survive. no development BA 1.0 0 f 0 d 0 d 0 d Survive, no development BA 2.0 0 f 0 d 0 d 0 d Survive, no development 2,4-D 0.1 37.6 e 0 d 0 d 0 d Global and heart shaped embryos 2,4-D 0.2 84.3 b 39.9 b 0 d 0 d Global, heart shaped, and cotyledon embryos 2,4-D 0.5 71.6 c 96.6 a 73.3 b 2.74b Global, heart shaped, and cotyledon embryos; white root 2,4-D 1.0 92.0 a 98.8 a 79.9 a 2.87a Global, heart shaped, and cotyledon embryos; white root 2,4-D 2.0 47.3 d 97.7 a 57.7 c 2.63c Yellow and some while 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 * Different letters within a column indicate significant differences at P<0.05 by Duncan’s multiple range tests. 6 3.1.1.2. Effect of single PGRs 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 PGRs on morphogenesis of petiole explant tTCL_C at photoperiod of 16 hours/day and in dark. PGRs (mg/l) Embryogeneis (%) Callogeneis (%) Root formation (%) Root number Remarks light Dark Contro l 0 0 e 0 e 0 d 0 d Necrosis 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 Black callus. Some white lateral root NAA 2.0 76.6 b 46.6 c 89.9 a 15.5 a Yellowish callus. A lot of white lateral root * Different letters within a column indicate significant differences at P<0.05 by Duncan’s multiple range tests. 3.1.1.3. Effect of single PGRs 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 PGRs on morphogenesis of lTCL_C petiole explant at photoperiod of 16 hours/day. PGRs (mg/l) Embryogenesis (%) Callogenesis (%) Root formation (%) Root number Remarks Control 0 0 e 0 e 0 f 0 c Necrosis TDZ 0.0 1 0 e 0 e 0 f 0 c Necrosis TDZ 0.0 5 0 e 0 e 0 f 0 c Necrosis 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 Few embryos. Black compact callus 2,4-D 0.5 59.6 c 89.9 b 79.9 a 4.7 ab Cluster of global, heart shaped and cotyledon embryos Yellow compact callus. Green elongated root 2,4-D 1.0 86.3 a 97.7 a 85.5 a 6.2 a Many global, heart, torpedo shaped and cotyledon embryos Brown callus. Yellowish roots 2,4-D 2.0 69.6 b 98.8 a 71 b 4.0 b Heart and global shaped embryos only at leaf edges Brown compact callus Few transparent white root 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 Large amount of callus. Purple embryo cluster NAA 2.0 84.0 a 96.6 a 37.7 c 4.0 b milky white heart and global shaped embryos. Little brown calli * Different letters within a column indicate significant differences at P<0.05 by Duncan’s multiple range tests. 8 Table 3.5. Effect of single PGRs on morphogenesis of lTCL_C petiole explant in dark. PGRs (mg/l) Embryogenesis (%) Callogenesis (%) Root formation (%) Root number Remarks Contr ol 0 0 c 0 e 0 g 0 f Necrosis 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 Embryogenic calli. Few yellow roots 2,4-D 0.2 0 c 67.7 c 35.5 de 0.91 d Large amount of yellow and white calli. White root 2,4-D 0.5 49.6 b 84.4 b 41.1 cd 0.51 e Few global embryos. Brown callus. Few white roots 2,4-D 1.0 69.6 a 94.4 a 46.6 c 1.87 c 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 NAA 1.0 0 c 45.5 d 61.1 b 6.09 b Little yellow callus. Milky white short roots. NAA 2.0 0 c 81 b 94.4a 19.2 a Little yellow callus Many milky white short roots. * Different letters within a column indicate significant differences at P<0.05 by Duncan’s multiple range tests. 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. PGRs (mg/l) Embryogenesis (%) Callogenesis (%) Root formation (%) Root number Remarks Contr ol 0 0 g 0 f 0 c 0 c Necrosis 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 Little white callus 2,4-D 0.2 31.0 f 87.7 b 0 c 0 c Large amount white callus. Few global shaped embryos. 2,4-D 0.5 57.3 d 89.9 b 0 c 0 c Large amount of brown calli. Cluster of global, and heart shaped embryos 2,4-D 1.0 79.6 b 97.7 a 0 c 0 c Large amount of dark brown calli. Global, heart shaped and cotyledon embryos. 2,4-D 2.0 91.0 a 88.8 b 0 c 0 c Large amount of dark brown calli. Many global, heart shaped and cotyledon embryos. NAA 0.1 0 g 0 f 0 c 0 c Necrosis NAA 0.2 71.0 c 0 f 0 c 0 c Embryo shaped global, heart, cotyledon, torpedo 10 NAA 0.5 70.6 c 0 f 0 c 0 c Global, heart, torpedo shaped and cotyledon embryos. NAA 1.0 48.6 e 44.4 e 74.4 b 5.09 b Global and heart shaped embryos. Few white roots NAA 2.0 47.3 e 67.7 d 83.3 a 9.24 a Global shaped and cotyledon embryos. Yellowish callus. Short white roots. * Different letters within a column indicate significant differences at P<0.05 by Duncan’s multiple range tests. Table 3.7. Effect of the combination of auxin and cytokinin on the morphogenesis of the rhizome explant tTCL_R in dark. PGRs (mg/l) Embryogenesis (%) Callogenesis (%) Root formation (%) Root number Remarks 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 Global shaped embryos. Yellowish callus. 2,4-D 0.2 84.0 a 61 b 24.4 c 0.93 b Global, heart shaped, and cotyledon embryos. Yellow callus 2,4-D 0.5 46.3 c 91 a 27.7 c 0.69 bc Few embryo 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 Explants turned yellow NAA 0.2 0 d 0 f 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 NAA 2.0 0 d 0 f 98.8 a 21.7 a Many white roots and lateral roots * Different letters within a column indicate significant differences at P<0.05 by Duncan’s multiple range tests. 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. PGRs (mg/l) Morphogenesis Remarks 2,4-D BA Callogenesis (%) 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 La