Summary of Philosophy doctoral dissertation on biology: Regeneration and micropropagation of panax Vietnamensis Ha et Grushv. using thin cell layer technology

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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.

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Nội dung Text: Summary of Philosophy doctoral dissertation on biology: 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
4 Table 3.1. Effect of single PGRs on morphogenesis of leaf explant tTCL_L in photoperiod of 16 hours/day Embryo Root Root PGRs Callogenes genesis formation numbe Remarks (mg/l) is (%) (%) (%) r Cont Necrosis 0 0e 0d 0e 0e rol TDZ 0.01 0e 0d 0e 0e Necrosis TDZ 0.05 0e 0d 0e 0e Necrosis TDZ 0.1 0e 0d 0e 0e Necrosis TDZ 0.2 0e 0d 0e 0e Necrosis TDZ 0.5 0e 0d 0e 0e Necrosis TDZ 1.0 0e 0d 0e 0e Necrosis BA 0.1 0e 0d 0e 0e Survive but no induction BA 0.2 0e 0d 0e 0e Survive but no induction BA 0.5 0e 0d 0e 0e Survive but no induction BA 1.0 0e 0d 0e 0e Survive but no induction BA 2.0 0e 0d 0e 0e Survive but no induction 2,4- Necrosis 0.1 0e 0d 0e 0e D 2,4- A few global embryos 0.2 43.3 d 0d 0e 0e D 2,4- A few global and heart 0.5 70.6 b 44.4 c 0e 0e D shaped embryos White, brown, and 2,4- 1.0 86.3 a 97.7 a 42.1 b 2.86 a compact callus. Embryos D cluster White, brown, and 2,4- 2.0 49.6 c 76.6 b 9.9 d 1.06 c compact callus. Few D embryos NA Necrosis 0.1 0e 0d 0e 0e A NA Necrosis 0.2 0e 0d 0e 0e A NA Necrosis 0.5 0e 0d 0e 0e A heart, global shaped NA 1.0 70.0 b 74.4 b 26.6 c 0.64 d embryos. Few short roots. A Dark brown callus Heart, global, cotyledon and torpedoe shaped NA 2.0 89.0 a 97.7 a 63.3 a 2.59 b embryos A Short white roots, dark brown callus * Different letters within a column indicate significant differences at P
5 Table 3.2. Effect of single PGRs on morphogenesis of leaf explant tTCL_L in dark conditions Root PGRs Embryogenesis Callogenesis Root formation Remarks (mg/l) (%) (%) number (%) Control 0 0f 0d 0d 0d Necrosis TDZ 0.01 0f 0d 0d 0d Necrosis TDZ 0.05 0f 0d 0d 0d Necrosis TDZ 0.1 0f 0d 0d 0d Necrosis TDZ 0.2 0f 0d 0d 0d Necrosis TDZ 0.5 0f 0d 0d 0d Necrosis TDZ 1.0 0f 0d 0d 0d Necrosis Survive. no BA 0.1 0f 0d 0d 0d development Survive. no BA 0.2 0f 0d 0d 0d development Survive. no BA 0.5 0f 0d 0d 0d development Survive, no BA 1.0 0f 0d 0d 0d development Survive, no BA 2.0 0f 0d 0d 0d development Global and 2,4-D 0.1 37.6 e 0d 0d 0d heart shaped embryos Global, heart shaped, and 2,4-D 0.2 84.3 b 39.9 b 0d 0d cotyledon embryos Global, heart shaped, and 2,4-D 0.5 71.6 c 96.6 a 73.3 b 2.74b cotyledon embryos; white root Global, heart shaped, and 2,4-D 1.0 92.0 a 98.8 a 79.9 a 2.87a cotyledon embryos; white root Yellow and some while 2,4-D 2.0 47.3 d 97.7 a 57.7 c 2.63c callus; few embryo, white root NAA 0.1 0f 0d 0d 0d Necrosis NAA 0.2 0f 0d 0d 0d Necrosis NAA 0.5 0f 0d 0d 0d Necrosis NAA 1.0 0f 0d 0d 0d Necrosis yellow callus NAA 2.0 0f 13.3 c 0d 0d at leaf egles * Different letters within a column indicate significant differences at P
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. Root Embryogeneis Callogeneis Root PGRs formation Remarks (%) (%) number (mg/l) (%) light Dark Contro Necrosis 0 0e 0e 0d 0d l TDZ 0.01 0e 0e 0d 0d Necrosis TDZ 0.05 0e 0e 0d 0d Necrosis TDZ 0.1 0e 0e 0d 0d Necrosis TDZ 0.2 0e 0e 0d 0d Necrosis TDZ 0.5 0e 0e 0d 0d Necrosis TDZ 1.0 0e 0e 0d 0d Necrosis BA 0.1 0e 0e 0d 0d Necrosis BA 0.2 0e 0e 0d 0d Necrosis BA 0.5 0e 0e 0d 0d Necrosis BA 1.0 0e 0e 0d 0d Necrosis BA 2.0 0e 0e 0d 0d Necrosis 2,4-D 0.1 0e 0e 0d 0d Necrosis 2,4-D 0.2 0e 0e 0d 0d Necrosis Soft yellowish 2,4-D 0.5 0e 83.3 a 0d 0d callus Soft yellowish 2,4-D 1.0 16.6 d 63.3 b 0d 0d callus 2,4-D 2.0 63.3 c 33.3 d 0d 0d Brown callus NAA 0.1 0e 0e 0d 0d Necrosis NAA 0.2 0e 0e 0d 0d Necrosis NAA 0.5 0e 0e 31.3 c 2.0 c White short root Black callus. NAA 1.0 86.6 a 0e 75.5 b 6.4 b Some white lateral root Yellowish callus. A lot of NAA 2.0 76.6 b 46.6 c 89.9 a 15.5 a white lateral root * Different letters within a column indicate significant differences at P
7 Table 3.4. Effect of single PGRs on morphogenesis of lTCL_C petiole explant at photoperiod of 16 hours/day. Root PGRs Embryogenesis Callogenesis Root formation Remarks (mg/l) (%) (%) number (%) Control 0 0e 0e 0f 0c Necrosis 0.0 Necrosis TDZ 0e 0e 0f 0c 1 0.0 Necrosis TDZ 0e 0e 0f 0c 5 TDZ 0.1 0e 0e 0f 0c Necrosis TDZ 0.2 0e 0e 0f 0c Necrosis TDZ 0.5 0e 0e 0f 0c Necrosis TDZ 1.0 0e 0e 0f 0c Necrosis BA 0.1 0e 0e 0f 0c Necrosis BA 0.2 0e 0e 0f 0c Necrosis BA 0.5 0e 0e 0f 0c Necrosis BA 1.0 0e 0e 0f 0c Necrosis BA 2.0 0e 0e 0f 0c Necrosis 2,4-D 0.1 0e 0e 0f 0c Necrosis Few embryos. Black 2,4-D 0.2 23 d 24.4 d 25.6 d 0.8 c compact callus Cluster of global, heart shaped and cotyledon embryos 2,4-D 0.5 59.6 c 89.9 b 79.9 a 4.7 ab Yellow compact callus. Green elongated root Many global, heart, torpedo shaped and 2,4-D 1.0 86.3 a 97.7 a 85.5 a 6.2 a cotyledon embryos Brown callus. Yellowish roots Heart and global shaped embryos only at leaf edges 2,4-D 2.0 69.6 b 98.8 a 71 b 4.0 b Brown compact callus Few transparent white root NAA 0.1 0e 0e 0f 0c Necrosis NAA 0.2 0e 0e 0f 0c Necrosis NAA 0.5 0e 0e 0f 0c Necrosis Large amount of NAA 1.0 58.6 c 84.4 c 10.6 e 3.1 b callus. Purple embryo cluster milky white heart and global shaped NAA 2.0 84.0 a 96.6 a 37.7 c 4.0 b embryos. Little brown calli * Different letters within a column indicate significant differences at P
8 Table 3.5. Effect of single PGRs on morphogenesis of lTCL_C petiole explant in dark. Root PGRs Embryogenesis Callogenesis Root formation Remarks (mg/l) (%) (%) number (%) Contr Necrosis 0 0c 0e 0g 0f ol TDZ 0.01 0c 0e 0g 0f Necrosis TDZ 0.05 0c 0e 0g 0f Necrosis TDZ 0.1 0c 0e 0g 0f Necrosis TDZ 0.2 0c 0e 0g 0f Necrosis TDZ 0.5 0c 0e 0g 0f Necrosis TDZ 1.0 0c 0e 0g 0f Necrosis BA 0.1 0c 0e 0g 0f Necrosis BA 0.2 0c 0e 0g 0f Necrosis BA 0.5 0c 0e 0g 0f Necrosis BA 1.0 0c 0e 0g 0f Necrosis BA 2.0 0c 0e 0g 0f Necrosis Embryogenic 2,4-D 0.1 0c 46.6 d 11.0 f 0.34 ef calli. Few yellow roots Large amount of yellow and 2,4-D 0.2 0c 67.7 c 35.5 de 0.91 d white calli. White root Few global embryos. 2,4-D 0.5 49.6 b 84.4 b 41.1 cd 0.51 e Brown callus. Few white roots White soft calli. Few embryos. 2,4-D 1.0 69.6 a 94.4 a 46.6 c 1.87 c Transparent white root 2,4-D 2.0 0c 0e 0g 0f Necrosis NAA 0.1 0c 0e 0g 0f Necrosis NAA 0.2 0c 0e 0g 0f Necrosis 31.1 e 0.94 d Few white NAA 0.5 0c 0e roots Little yellow callus. NAA 1.0 0c 45.5 d 61.1 b 6.09 b Milky white short roots. Little yellow callus NAA 2.0 0c 81 b 94.4a 19.2 a Many milky white short roots. * Different letters within a column indicate significant differences at P
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. Root PGRs Embryogenesis Callogenesis Root formation Remarks (mg/l) (%) (%) number (%) Contr Necrosis 0 0g 0f 0c 0c ol TDZ 0.01 0g 0f 0c 0c Necrosis TDZ 0.05 0g 0f 0c 0c Necrosis TDZ 0.1 0g 0f 0c 0c Necrosis TDZ 0.2 0g 0f 0c 0c Necrosis TDZ 0.5 0g 0f 0c 0c Necrosis TDZ 1.0 0g 0f 0c 0c Necrosis BA 0.1 0g 0f 0c 0c Necrosis BA 0.2 0g 0f 0c 0c Necrosis BA 0.5 0g 0f 0c 0c Necrosis BA 1.0 0g 0f 0c 0c Necrosis BA 2.0 0g 0f 0c 0c Necrosis 75.5 c Little white 2,4-D 0.1 0g 0c 0c callus Large amount white callus. Few 2,4-D 0.2 31.0 f 87.7 b 0c 0c global shaped embryos. Large amount of brown calli. 2,4-D 0.5 57.3 d 89.9 b 0c 0c Cluster of global, and heart shaped embryos Large amount of dark brown calli. Global, heart 2,4-D 1.0 79.6 b 97.7 a 0c 0c shaped and cotyledon embryos. Large amount of dark brown calli. Many global, 2,4-D 2.0 91.0 a 88.8 b 0c 0c heart shaped and cotyledon embryos. NAA 0.1 0g 0f 0c 0c Necrosis Embryo shaped global, heart, NAA 0.2 71.0 c 0f 0c 0c cotyledon, torpedo
10 Global, heart, torpedo shaped NAA 0.5 70.6 c 0f 0c 0c and cotyledon embryos. Global and heart NAA 1.0 48.6 e 44.4 e 74.4 b 5.09 b shaped embryos. Few white roots Global shaped and cotyledon NAA 2.0 47.3 e 67.7 d 83.3 a 9.24 a embryos. Yellowish callus. Short white roots. * Different letters within a column indicate significant differences at P
11 NAA 0.5 0d 24.4 e 48.8 b 0.59 bc Few roots 52.2 b 1.37 b Few yellow NAA 1.0 0d 0f short roots Many white NAA 2.0 0d 0f 98.8 a 21.7 a roots and lateral roots * Different letters within a column indicate significant differences at P
12 Little brownish yellow and transparent 0.2 1.0 80.0 c 0c compact callus 0.5 1.0 93.3 b 0c White and milky white soft callus 2.0 1.0 93.3 b 0c White and brownish red soft callus * Different letters within a column indicate significant differences at P
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. PGRs Morphogenesi (mg/l) s Remarks Callogenesis NAA BA (%) 1.0 0.1 13.3 c Little green compact callus 1.0 0.2 0d Necrosis 1.0 0.5 0d Necrosis 1.0 1.0 33.3 b Little brown compact callus 1.0 2.0 0d Survival but no development 0.1 1.0 0d Necrosis 0.2 1.0 0d Necrosis 0.5 1.0 0d Necrosis 2.0 1.0 60 a Little green compact callus * Different letters within a column indicate significant differences at P
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 PGRs Morphogenesis (mg/l) Remarks Callogenesis 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 * Different letters within a column indicate significant differences at P
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 0c Necrosis * Different letters within a column indicate significant differences at P
16 Table 3.19. Effect of the combination of NAA and BA on the morphogenesis of the tTCL_C petiole explant in dark. PGRs Morphogenesis (mg/l) Remarks Callogenesis 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 0b 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 * Different letters within a column indicate significant differences at P
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 PGRs Morphogenesis (mg/l) Shoot Remarks 2,4- Callogen BA formation D esis (%) (%) 1.0 0.1 100 a 0b Brownish yellow, and milky white soft callus 1.0 0.2 100 a 21.0a Large amount of milky white soft callus 1.0 0.5 100 a 0b Milky white soft callus 1.0 1.0 100 a 0b Little yellow and milky white soft callus 1.0 2.0 100 a 0b Light yellow soft callus 0.1 1.0 46.7 d 0b Little brownish red compact callus Little yellow and brown compact callus on surface and two 0.2 1.0 80.0 c 0b cut explants Milk white soft callus on explant surface and two cut 0.5 1.0 93.3 b 0b surfaces 2.0 1.0 100 a 0b Little yellow and brown soft callus * Different letters within a column indicate significant differences at P
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 PGRs (mg/l) Morphogenesis Remarks 2,4-D TDZ Callogenesis (%) 1.0 0.01 93.3 b Milky white, and brown soft callus on one cut surface Little yellow and milky white soft callus on one cut 1.0 0.05 100 a surface Large amount of transparent and milky white soft callus 1.0 0.1 100 a on two cut surfaces 1.0 0.2 100 a Milky white and brownish yellow soft callus Large amount of yellow and milky white soft callus on 1.0 0.5 93.3 b one cut surface Little milky white and yellow soft callus on two cut 1.0 1.0 86.6 c surfaces 0.1 0.2 0e Survival but no development White and brownish yellow soft callus on explant 0.2 0.2 80.0 d surface and two cut surfaces White and brownish yellow soft callus on explant 0.5 0.2 100 a surface and two cut surfaces 2.0 0.2 86.6 c Little brownish yellow soft callus on one cut surfaces * Different letters within a column indicate significant differences at P