HUE UNIVERSITY Hue University of Sciences NGUYEN THI NHAT LINH

Transcription

1 HUE UNIVERSITY Hue University of Sienes NGUYEN THI NHAT LINH EFFECT OF CULTURE CONDITIONS AND ELICITORS ON THE GROWTH AND SAPONIN ACCUMULATION IN SECONDARY ROOT OF NGOC LINH GINSENG (Panax vietnamensis Ha et Grushv.) CULTURED IN VITRO SUMMARY OF PhD. THESIS - PLANT PHYSIOLOGY HUE - 217

2 The researh is ompleted at: TAY NGUYEN INSTITUTE FOR SCIENTIFIC RESEARCH Instrutor: Prof.Dr Duong Tan Nhut Prof.Dr Nguyen Hoang Lo Reviewer 1: Reviewer 2: Phản biện 3: The thesis was defended before Thesis Examination Committee at Hue University level at: At...time...date...month...year... The thesis an be found at: - National Library of Vietnam; - Learning Resoure enter Hue University; - Library Hue University of Sienes Hue University.

3 1 INTRODUCTION Vietnamese ginseng, Ngo Linh Ginseng (Panax vietnamensis Ha et Grushv.) is an endemi ginseng speial of Vietnam ginseng. This ginseng has been studied and showed many pharmaeutial ingridients than other speials of same genus, inluding 26 saponins like Amerian ginseng and Korean ginseng; besides, there was found more than 2 other saponins in Ngo Linh ginseng. These saponins help health improvement, vitality enhanement, blood pressure ontroll, antioxidants, aner preventation, antiaging, immune stimulation, treatment of malaria, depression, liver, kidney and diabetes... This valuable ginseng has been very sare and ranked in the red list book that needs to be preserved, but it still over-exploited in nature. Moreover, using ompounds of Ngo Linh ginseng in pharmaeutial industry was still diffiulty. In vitro root ulture is onsidered as potential alternative method to havest a large biomass in a shortime. In vitro Ngo Linh ginseng root was optimally grown when it was ulture in the pertinent medium and onditions. Although the inrease of root biomass is important, the saponin aumulation is more nesessary. For the reason that the largest root biomass having the highest saponin ould be used as a raw material for the prodution of funtional foods, even to produe drugs. In fat, ulturing Ngo Linh ginseng by in vitro roots, the root yeild developed quikly, but saponin ontent is lower than the wild ginseng roots. Eliitors have the great potential in enhaning the saponin aumulation in Ngo Linh ginseng ulture, so the researh on the appliation of eliitors into the ginseng lateral root ulture is very neessary. Therefore, the thesis Improve seondary root ulture of

4 2 and investigate eliitor on saporin aumulation of Ngo Linh ginseng (Panax vietnamensis Ha et Grushv.) was studied to not only improve the formation and growth of lateral roots (s) from in vitro adventitious roots (ARs) by optimizing ulture medium and ulture onditions, but also enhaning the aumulation of three saponin (MR2, Rb1 and Rg1) in Ngo Linh ginseng via the effetiveness of eliitors. Objetives of thesis: Optimazize the in vitro Ngo Linh ginseng lateral root ulture and multipliate the root biomass is more stable and higher in bioreator system. Determine and evalute the effet of different types, and onentrations of five eliitors (SA, JA, ABA, CHN và YE) on produing saponin ompounds. Next, ombine in various types of eliitors to enhane effetiveness of the saponin aumulation, and determine the best eliitation time to develop the highest saponin in s of Ngo Linh từ ARs in vitro. Sienifi signifiane: Providing sienifi valuable data about root morphology, multipliation of root biomass and enhane saponin ontent in ulturing in vitro s of Ngo Linh ginseng. In addition, the thesis is also a useful referene for the study and teahing in metabolism field, plant physiology and tissue ulture. Pratial signifiane: This study had established the ulture medium and ulture ondition for the development of in vitro Ngo Linh ginseng s ulture in short time. In addition, this study had determined the best of type, onentration and eliitation time for aumulating saponin ompounds (MR2, Rg1 và Rb1) in s. The results suggest that the treatment is optimized for inreasing the growth and saponin ontent of s; thus, the ginseng saponin prodution ould be sale-up via root ulture.

5 3 CHAPTER 1. LITERATURE REVIEW 1. NGOC LINH GINSENG Ngo Linh ginseng (Panax vietnamensis Ha et Grushv.) was found at Ngo Linh mountain, DaTo distrit, Kon Tum provine. This ginseng has been studied and showed that it has many pharmaeutial ompounds than other Panax genus. 2. ADVENTITIOUS ROOT (AR) and LATERAL ROOT () CULTURE s originate from periyle ells of existing roots like the PR, earlier s, or ARs. Depending on the speies, they may derive from periyle ells adjaent to xylem pole or to phloem pole ells of diot plant suhs as Ngo Linh gingseng. In monoot plant, the endodermal ells also ontribute to development and produe the epidermis and the root ap, whereas the periyle ells are the soure of all remaining tissues. The different stages of initiation and development have been tarting from the first ell division in the periyle ell up to the emerging primordium, seven stages have been identified that orrespond to different steps in the aquisition of ell identity and tissue organization. In ontrast to s, ARs develop from different tissues and onsequently from different ell types. A onserved feature is that ARs always develop from ells neighboring vasular tissues, whether they are part of the plant s development program or are artifiially indued by wounding or hormone appliations. Often, ell division had already started and/or small AR primordia were already formed.

6 4 3. SAPONIN COMPOUNDS Saponins are naturally ourring surfae-ative glyosides. It is a diverse group of terpenoids haraterized by their struture. Saponins are omposed of a steroidal or triterpenoid aglyon and one or more sugar hains. A plant might ontain several dozen saponins with losely related strutures. The latest researh of pharmaologial ompounds showed that saponins of Ngo Linh ginseng have over 6 types; the most important group of the ginseng seondary metabolites is the saponin triterpene whih the main representatives are MR2, Rb1 and Rg1 in the dammaran group. Triterpene saponins are omplex strutures, so the saponin synthesis is not eonomially feasible for produing on a large sale, and the wild ginseng soures are extremely rare. 4. ELICITOR and ELICITATION The term eliitor is very general and refers to hemials from various soures, bioti or abioti, as well as physial fators, that an trigger a response in living organisms resulting in aumulation of seondary metabolites. Eliitation is the indued or enhaned biosynthesis of metabolites due to addition of trae amounts of eliitors. So far, studies on the appliation of eliitors to in vitro ulture of Ngo Linh ginseng to improve the ontent of saponins are very limited. Duong Tan Nhut et al., (212) initially assessed the effet of MeJA on the saponin aumulation in the ginseng alli. Trinh Thi Huong (217) has also begun to study the effets of MeJA, SA and ABA to enhane saponin ompounds in the transgeni root ulture.

7 5 CHAPTER 2. MATERIALS AND METHODS 1. ROOT EXPLANTS In vitro adventitious root lusters of P. vietnamensis, whih were indued from leaves (1 1 m) and grown for 4 days on SH medium (Shenk and Hildebrandt 1972) supplemented with 5 mg/l IBA, 3 g/l surose, and 8 g/l agar, were used as the soure of explants. 2. RESEARCH CONTENTS 2.1. Optimal explant resoure and medium ulture for Ngo Linh ginseng s growth from in vitro ARs Investigation of the effet of the in vitro ARs uttings on the formation and growth of Ngo Linh ginseng s Investigation of the effet of auxin on the formation and growth of Ngo Linh ginseng from in vitro ARs Investigating the effet of the ombination of auxin and ytokinin on the formation and growth of Ngo Linh ginseng s Investigation of the influene of mineral medium on the formation and growth of Ngo Linh ginseng s Investigating the effets of NH + 4 /NO - 3 in MS medium on the formation and growth of Ngo Linh ginseng s Investigation of the effets of arbohydrates on the formation and growth of Ngo Linh ginseng s Investigation of the effet of ulture medium on the formation and growth of Ngo Linh ginseng s 2.2. Optimized unultured root ulture onditions of Ngo Linh ginseng to enhane in vitro formation and saponin aumulation Investigation of the effet of ulture time on the growth and aumulation of saponins of Ngo Linh ginseng Investigation of the effet of ulture temperature on the growth

8 6 and aumulation of saponins of Ngo Linh ginseng s Investigation of the effet of ph on saponin growth and aumulation of Ngo Linh ginseng from in vitro ARs Investigation of the effet of dark and light ulture on the growth of Ngo Linh ginseng s Investigation of the effet of ulture volume on the formation and growth of Ngo Linh ginseng from ultured in vitro ARs Investigation of the effet of ulture system on the growth and aumulation of saponins of Ngo Linh ginseng s 2.3. Optimal saponin aumulation of s formed from the ARs of Ngo Linh ginseng via the effet of eliitors Investigation of the effets of hitosan on the growth and saponin aumulation of Ngo Linh ginseng from in vitro ARs Investigation of the effet of yeast extrat on the growth and saponin aumulation of Ngo Linh ginseng s The effet of absisi aid on the growth and saponin aumulation of Ngo Linh ginseng s Investigation of the effet of saliyli aid on the growth and saponin aumulation of Ngo Linh ginseng s Investigation of the effets of jasmoni aid on the growth and saponins aumulation of Ngo Linh ginseng s Investigation on the ombined effet of exogenous eliitor and endogenous eliitor on the growth and saponin aumulation of Ngo Linh in vitro Investigation on the effet of eliitation time on the growth and saponin aumulation of in vitro Ngo Linh Determination of growth rate, dry matter perentage, formation rate and proliferation of the ulture

9 Culture media The ulture was maintained on the modified MS medium (Murashige and Skoog 1962) of whih the ratios of NH + 4 /NO 3 were 7.19:18. mm/mm supplemented with 7. mg/l IBA,.5 mg/l BA, and 3 g/l surose. The medium was adjusted ph to 5.8 prior to autolaving, and the medium was autolaved for 3 min at 121 C and 1 atm Culture onditions The in vitro Ngo Linh AR ultures were maintained in the darkness abinet with temperature ± 2 C and relative humidity 55-6%. Exept for solid ulture, the root ulture was shaken on the Innova 2 plantform shaker (Hermle, Germany) at rpm. 3. SAPONIN ANALYSIS 3.1. Thin layer hromatography on the saponin analysis 3.2. High performane liquid hromatography 3.4. Determination method of saponin produtivity of ginseng s 4. CYTOLOGY METHODS 5. QUANTITATIVE DATA ANALYSIS USING SPSS 2.

10 8 CHAPTER 3: RESULTS 1. OPTIMIZE NGOC LINH GINSENG CULTURE FROM IN VITRO ARS 1.1. Optimal explant resoure and medium ulture for Ngo Linh ginseng s growth from in vitro ARs Effet of the in vitro ARs uttings on the formation and growth of Ngo Linh ginseng s The results of adventitious root (ARs) influent study on the formation and growth of in vitro Ngo Linh ginseng the lateral root () showed in Table 3.1. The soure from the petiole pattern has the size of 2 m, no separate the pattern is the most suitable for the formation and growth of in vitro Ngo Linh ginseng s. Table 3.1. Effet of the in vitro ARs uttings on the formation and growth of Ngo Linh ginseng s AR derived from Leaf dis Petiole Rhizome SM C1 C2 C1 C2 C1 C2 AR lenght (m) formation rate (%) number lenght (m) FW e *.33 h.37 f g d 2. efg.83 f f b 2.33 ef 2. e a b 73. b d 1.33 fgh 1.67 e 3.33 g d 1. b 5.23 a e b 3. e 3.13 d d a a 5.6 a 78. a d.67 gh.67 f f d 4.3 b 51. e b de d b a 5. a b * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, SM: sample utting, C1: ut on AR and removed regeneration explant, C2: no removed regeneration explant.

11 Effet of auxin on the formation and growth of Ngo Linh ginseng from in vitro ARs. Table 3.2. Effet of auxin on the formation and growth of Ngo Linh ginseng from in vitro ARs. NAA IBA Auxin (mg/l) formation rate (%) number lenght (m) FW DW. f *. i. f h 1.33 i 3 6. e 7.67 f.83 e 4.2 g 2.7 h d f 4. fg e a d 5.7 d de 7.33 f 4.67 b 65. d 5. de e 1.77 d e 5.67 d ab 16. a 5.17 b b ab a 6. a a a a b 4.6 b b b d 5.33 h 1.3 e 32. g 1.33 i 5 9. b 6.33 g 1.83 d f 3.17 gh IAA b d e 4.67 ef 9 9. b d d 6.83 * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight Effet of the ombination of auxin and ytokinin on the formation and growth of Ngo Linh ginseng s Table 3.3. Effet of the ombination of IBA and ytokinin on the formation and growth of Ngo Linh ginseng s IBA (mg/l) Cytokinin (mg/l) formation rate (%) number length (m) FW DW a * a 5. a 155. de 14. de b de 85. g 6. hi Kin d 4. de.75 ef 121. f def f 2.75 ef.45 fgh 229. b 17. b 2.. g 1. fg.2 gh 7. g 5.98 hi ab 1. b 1.95 b 1. f 7.75 h 7, a 12. a 2.23 b 26. a 21. a BA b 4.75 d 1. d 132. ef 12. efg ef 1.75 fg.7 ef 164. d fg ab 4. de f 1.23 g TDZ de 2.75 ef.85 def 233. ab 15. d.1 3. g 1. fg.65 efg b.2 1. h.75 g.13 h 6. g 5. i * Different letters indiate signifiant differene (P <.5) with Dunan s test.

12 Effet of mineral medium on the formation and growth of Ngo Linh ginseng s Table 3.4. Effet of mineral medium on the formation and growth of Ngo Linh ginseng s Medium formation rate (%) number length (m) FW DW SH 78.7 b* 16.5 b 3.63 b s and ARs b s and ARs in white and slender. a Big s and MS 92.5 a 18.3 a 4. a a 31.3 Yellowing thik ARs B ab b 21.8 b nerosis s and Yellowing or ARs * Different letters indiate signifiant differene (P <.5) with Dunan s test Effet of NH 4 + /NO 3 - and growth of Ngo Linh ginseng s rate in MS medium on the formation Table 3.5. Effet of NH 4 + /NO 3 - rate in MS medium on the formation and growth of Ngo Linh ginseng s NH + - FW DW 4 /NO 3 number length (m) / * 4.33 b b b 1/ a 6.57 a 396. a a 1/ b 3. d 281. b /2 13. d 2.67 d d 2/ 4. f 2.87 d d 5.67 f 2/ b 2.6 d b b 2/3 11. e 4.67 b d 9.27 e * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight.

13 Number and lenght of s (m) Dry weight Graph 3.1. Comparisons between the modified medium had the best NH 4 + /NO 3 - ratio ompared to other ulture media. * Different letters on eah olumn indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight Effets of arbohydrates on the formation and growth of Ngo Linh ginseng s Table 3.6. Effets of arbohydrates on the formation and growth of Ngo Linh ginseng s Carbohydrate (g/l) D- Gluose Surose e number Root length (m) DW a a a b b d b b FW e DW d 1/2 MS 1/2 SH 3/4 MS 3/4 SH MS MSCB SH Culture medium Dry matter rate (%) s and ARs e * 3.33 hi 6.42 d Nerosis d 15. gh 9.82 b white, long and slightly branh d g 8.89 bd White, very long, slightly branh e 1.96 ab White, very long, big, less branh b bd 8.99 bd White, very long, big, less branh b 67.4 a 13.3 a White, very long, big, less branh f 8.85 bd White, very long, big, less branh a b 8.64 bd White, very long, big, high branh b 56. b 9.79 ab White, very long, big, high branh b de 9.16 bd White, very long, big, high branh b de 7.8 d White, very long, big, high branh e 9.hi 8.22 bd Yellow, less branhed or prolonged d gh 9.54 b Yellow, less branhed Maltose d f 9.76 b Yellow and nerosis, less branhed e 7.67 hi 7.37 d Yellow and nerosis, less branhed e 4.33 hi 6.94 d Yellow and nerosis, less branhed *Different letters indiate signifiant differene (P <.5) with Dunan s test. f d b a a

14 Effet of ulture substrate on the formation and growth of Ngo Linh ginseng s Table 3.7. Effet of ulture substrate on the formation and growth of Ngo Linh ginseng s ulture formation FW substrate rate (%) number length (m) DW Agar a* 2.7 a a a Gelrite b.94 b a a Cotton b 77.2 b 8.27 b * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight Effet of ulture period on the formation and growth of Ngo Linh ginseng s from in vitro AR ulture Effet of ulture period on the growth of Ngo Linh ginseng 3 1 s from in vitro ARs ulture f d e d e e d Graph 3.2. Effet of ulture period on the formation and growth of Ngo Linh ginseng s from in vitro ARs ulture The effet of temperature on the formation and growth of the Ngo Linh ginseng s ulture from in vitro ARs When the survey affets the ulture temperature from C on the formation and growth of Ngo Linh ginseng ARs, a a b b a ab b Culture period (days) DW FW Root number Poly. (FW ) b

15 13 the result after 56 days showed that 22 C was suitable for the Ngo Linh ginseng formation and growth. Table 3.8. Effet of temperature on the s growth from in vitro Ngo Linh ginseng ARs Temperature ( C) formation rate (%) number length (m) FW DW 22 a* a 1. a a 8.33 a a b.73 b 12. b 6.67 b 28 a * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight Effet of ph on the formation and growth of Ngo Linh ginseng from in vitro ARs. Table 3.9. Effet of ph on formation and growth of Ngo Linh ginseng from in vitro ARs. ph formation rate FW DW (%) number length (m) 4.3 a* b a b 1.7 b b b 5.3 a 56. a 1.37 a a 2.67 a 5.8 a 3. b 1. b b a b * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight Effet of dark and light ulture on the formation and growth of Ngo Linh ginseng Table 3.1. Effet of dark and light ulture period on the growth of Ngo Linh ginseng s Treatment formation rate (%) number length (m) FW DW % dark 94. a* a 5.66 a a b 8% dark a 19. a 5. a 369. a a % dark b 8.67 b 2.83 b b % light d * Different letters indiate signifiant differene (P <.5) with Dunan s test. 8% dark: 8% frist ulture day in dark; % dark: % frist ulture day in dark; % dark: % frist ulture day in dark; % light: % frist ulture day under 16h/day.

16 Effet of ulture volume on the growth of Ngo Linh ginseng from ultured in vitro ARs Table Effet of ulture volume on the growth of Ngo Linh ginseng from ultured in vitro ARs Volume formation (ml) number rate (%) length (m) FW DW d * 2.12 b a 3.18 a a 45.6 a 38.8 b 2.84 a b 18.2 b b * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight Effet of ulture system on the growth of Ngo Linh ginseng s from in vitro AR ulture Table Effet of ulture system on the growth and aumulation of saponins of Ngo Linh ginseng s Culture system FW (g) DW (g) Proliferation rate (times) Dry matter rate (%) Aeration flask 3 L a* 6.74 a 9.71 a 9. a Shaking flask 3 L b Bubble boreator 3 L 51.3 b 4.63 b 6.84 b 9.3 ab * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight.

17 15 2. OPTIMAL SAPONIN ACCUMULATION OF NGOC LINH GINSENG S FROM AR CULTURE VIA EFFECTIVE OF ELICITORS 2.1. Effet of ulture onditions on the saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture Effet of ulture period on the saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture Table Effet of ulture period on the saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture Culture period Saponin ontent ( )* NS (days) Rg1 Rb1 MR2 Total of 3 saponin (mg/l) 14. d. d. d. d. e b.74 d d 42.6 b.3 a b 2.47 b 2.29 a 7.6 b.2 b 3.13 a 3.21 a 1.89 b 84.8 a.2 b 3.26 a 3.36 a 1.83 b * Averagement of 3 saponin HLPC analysis per 1 g dry matter; NS: Produtivity of 3 saponins on total of dry biomass Effet of temprerature on the saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture Table Effet of temprerature on the saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture Saponin ontent ( )* Total of 3 Rg1 Rb1 MR2 saponin Temprerature ( C) NS a.64 a 1.4 a.4 a.7 b.73 b.35 b 1.12 b.23 b 28.7 b b * Averagement of 3 saponin HLPC analysis per 1 g dry matter; NS: Produtivity of 3 saponins on total of dry biomass.

18 Effet of ph on the saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture Table Effet of ph on the saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture ph Saponin ontent ( )* Rg1 Rb1 MR2 Tổng 3 saponin NS b b b b.2 ab 1.62 b 1.68 b 1.16 a b.2 a 2.45 a 2.51 a.95 ab a.2 b 1.61 b 1.67 b.45 * Averagement of 3 saponin HLPC analysis per 1 g dry matter; NS: Produtivity of 3 saponins on total of dry biomass Effet of ulture system on the saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture Table Effet of ulture system on the saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture Culture system Saponin ontent ( )* Rg1 Rb1 MR2 Tổng 3 saponin NS Aeration flask 3 L b 4.15 b.93 b Shaking flask 3 L.35 a 1.9 a b.3 Bubble boreator 3 L. ab 1.41 ab 6.86 a 8.52 a 1.31 a * Averagement of 3 saponin HLPC analysis per 1 g dry matter; NS: Produtivity of 3 saponins on total of dry biomass.

19 Effet of eliitors on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture Effet of hitosan on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture Table Effet of hitosan (CHN) on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture Content (% per 1 g DW) CHN FW DW (mg/l) Rg1 Rb1 MR2 Total 3 saponin a* a.339 d.144 d d e.466 a.164 e.89 d e e a.142 d.1 d 2.3 a d 2.32 d.676 b.328 b.433 a 1.43 b b 29. b b 1.2 * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight Effet of yeast extrat (YE) on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs. Table Effet of YE on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture YE (mg/l) FW DW Content (% per 1 g DW) Rg1 Rb1 MR2 Total 3 saponin * d.88 d a 51.a.2 b.1 a b b 47. b.534 b. b.743 b 1.42 b 1.3 d.3 d a.244 a.786 b 3.51 a e e.544 b.6 a.842 a 1.67 * Different letters indiate signifiant differene (P <.5) with Dunan s test.

20 Effet of absisi aid (ABA) on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs Table Effet of absisi aid on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture ABA FW DW Content (% per 1 g DW) (µg/l) Rg1 Rb1 MR2 Total 3 saponin a* a.339 d.144 d.395 d.88 e b b b 1.96 d b d 8. d 1.6 a.219 b a 3.37 a 76.3 d 7.58 d a a 2.65 b ** Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight Effet of salyisi aid (SA) on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs Table 3.2. Effet of salyisi aid (SA) on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs SA (µg/l) FW DW Content (% per 1 g DW) Rg1 Rb1 MR2 Total 3 saponin a* a.339 d.144 d d b a.675 a.248 b.867 a 1.79 a b a.599 b 1.44 b d b b d b * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight Effet of jasmoni aid (JA) on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs Table Effet of jasmoni aid (JA) on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture JA FW DW Content (% per 1 g DW) (µg/l) Rg1 Rb1 MR2 Total 3 saponin a* a.339 e.144 e.395 e.88 d b b a a.718 b 1.32 d 5.24 b d 8.59 d b b 5.57 a d 7.59 d d.41 d a 5.21 b * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight.

21 Tổng 3 saponin (%) Rb1 (%) MR2 (%) Rg1 (%) Năng suất tổng hợp 3 saponin (mg/l) Comparation of the effets of solitary eliitors on the growth and saponin aumulation of Ngo Linh s f hij hij ij k fg e f fgh Graph 3.3. Effet of solitary eliitors in saponin aumulation of Ngo Linh s from in vitro ARs ulture ĐC CHN (mg/l) YE(mg/L) ABA (µg/l) SA (µg/l) JA (µg/l) ghi ĐC ABA (µg/l) CHN (mg/l) SA (µg/l) YE (mg/l) JA (µg/l) l l d a j b b e fg 1 1 ĐC CHN (mg/l) YE(mg/L) ABA (µg/l) SA (µg/l) JA (µg/l) ĐC CHN (mg/l) YE(mg/L) ABA (µg/l) SA (µg/l) JA (µg/l) ĐC CHN (mg/l) YE(mg/L) ABA (µg/l) SA (µg/l) JA (µg/l) Graph 3.4. Comparation of effets of solitary eliitors in saponin aumulation of Ngo Linh s from in vitro ARs ulture

22 Effet of the simultaneous ombination of SA and YE on the growth and saponin aumulation of Ngo Linh ginseng s ulture from in vitro ARs Table Effet of simultaneous ombination of SA and YE on the growth and saponin aumulation of Ngo Linh ginseng s ultured from in vitro ARs SA (µg/l) YE (mg/l) FW (mg/l) DW (mg/l) Growth index % DW a a a 9.23 bde 44. a b 16.6 a 7.69 def d e bde de h 1.26 de 7.73 def ghi 19. ij 7.76 ghi 8.68 bde 25. hijk 17.6 km 7.2 hijk 8.32 bd e ijk mn 6.29 ijk 8.61 bde ghij ijk 7.68 ghij 9.78 b 5. efg f 9.2 efg 9.68 b ef 34. b 9.68 ef a 174. k mn 5.97 k 9.4 bd ef fg 9.65 ef 8.89 bde fgh 23. gh 8.63 fgh 9.57 bd 283. de 22. h 1.32 de 7.77 def d. f 11.8 d 7.82 def ijk 16. mn 6.93 ijk 8.7 def de ijk 1.55 de 6.35 f b b 8.8 def b 3.11 d b 7.44 ef 1 5. efg 2. i 9.2 efg 7.86 def jk 15. n 6.15 jk 8.39 bde b b b 8.61 bde d 28. e d 8.99 bde de fg 1.61 de 8.15 def hijk ij 7.47 hijk 9.13 bde 192. ijk 18.6 jk 6.69 ijk 9.39b de * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight.

23 Năng suất tổng hợp 3 saponin (mg/l) ĐC ĐC Rg1 (%/g KLK) ĐC MR2 (%/g KLK) ĐC Rb1 (%/gklk) a b d de d e efg h g j i fgh f fg hi h h g j j jk kl l k k 1 YE (mg/l) SA (µg/l) i d b e ef d ij ijk h j k h f bd de e de e ef d e ij f g a YE (mg/l) mn e d 1 o fg m n l l o n j f g ijk ij k i ij a lm gh h b l SA (µg/l) YE (mg/l) SA (µg/l) a b b b d d j i efg g hi fgh e d e fg f de h i l l k jk YE (mg/l) 1 SA (µg/l) Graph 3.5. Effet of simultaneous ombination of SA and YE on the saponin aumulation of Ngo Linh ginseng s from in vitro ARs

24 NS (mg/l) FR Rg1 (%/g DW) MR2 (%/g DW) NS (mg/l) Rb1 (%/g DW) ,26 b 4,4,13 28, d 373,4 b 374,5 b,8 e,1d ,5 a T T1 T2 T3 T4,29 a Graph 3.6. Effet of simultaneous ombination of µg/l SA and mg/l YE ompared with 1 mg/l YE on the saponin aumulation of Ngo Linh ginseng s ulture from in vitro ARs Effet of eliitation time on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs Table Effet of eliitation time on the growth and saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture Thời gian (ngày) 9.15 FW DW mg/l YE + µg/l SA 1 mg/l YE.2. mg/l YE + µg/l SA 1 mg/l YE Content (% per 1 g DW) Content (µl/l) Rb1 MR2 Rg1 Total Rb1 MR2 Rg1 Total d.7858 a.245 a 1. a e b 24.5 a b b.42 e.667 b a b a b 33.2 b.3112 a.2366 d b 567. a d 17.7 b b b b.12 e.51 d.328 d 45.3 b e 9.37 d d a 64. a b d e * Different letters indiate signifiant differene (P <.5) with Dunan s test. FW: Fresh weight, DW: Dry weight..24 mg/l YE + µg/l SA 1 mg/l YE mg/l YE + µg/l SA 1 mg/l YE Graph 3.7. Eliitation time on the saponin aumulation of Ngo Linh ginseng s from in vitro ARs ulture

25 23 CONCLUTION The results showed that, 1. The length of the Ngo Linh ginseng AR explants whih regenerated from the petioles, were optimal for the s formation and growth about 2 m, but were not exised ompletely the petiole patterns. 2. The optimal medium for developing s is MS medium modified with 1/2 NH + 4 /NO - 3, supplemented with 7 mg/l IBA,.5 mg/l BA and 3 g/l surose. 3. The most appropriate ulture onditions for the formation, growth and saponin aumulation of the gingseng s from in vitro ARs were ultured in 56 days with the first 8% of ulture period in dark, and maintained at a temperature of 22ºC and ph 5.3. In bottle ml, mg ARs developed the highest of s and three-saponin-ontents (MR2, Rb1 and Rg1) in 3 ml ulture medium, respetively using 8 g explants in 1.5 L medium ontained in balloon-type bubble bioreator 3 L. 4. Eliitors enhaned signifiantly the aumulation of MR2, Rb1 and Rg1, but inhibited the lateral roots growth of Ngo Linh ginseng. The optimal solitary eliitor treatment for the aumulation Rb1 is µg/l JA, Rg1 is µg/l JA, and MR2 is µg/l JA, but JA and ABA inhibited the growth more than SA, YE and CHN. Produtivity of total saponins is highest at 1 mg/l YE. 5. The ombination of YE and SA showed effetive lower than the solitary eliitor treatment (1 mg/l YE). 6. The eliitation time of 1 mg/l YE treatment at the 4 th ulture day is optimal for saponin aumulation in lateral root ulture

26 24 SUGGESTIONS In the limited about eonomi onditions and study time, thesis was only reported in the speified ontents. Thus, to improve ompletely researh, we are reommended to ontinue the researh of ontent after: Investigate eliitation time of JA, ABA or SA on ARs and s after harvesting the Ngo Linh large root biomass. Study on effets of sequential treatment of various eliitors in ulture of Ngo Linh ginseng through the sequential expression of enzymes that atalyze the synthesis of saponins in the triterpenoid pathway. Study on effets of the eliitors on the speifi genes to aumulate saponins in Ngo Linh ginseng, for produing the desired saponins. Sale up ulture for ginseng saponin prodution to response a soure of raw materials for pharmaeutial and food industries.

27 LISTS OF PUBLICATIONS OF THE AUTHOR 1. Nguyen Thi Nhat Linh, Nguyen Hoang Lo, Duong Tan Nhut (218). Appliation of eliitor for prodution of saponins from in vitro Panax ultures. Journal of Biotehnogy, 15(2): Nguyen Thi Nhat Linh, Hoang Thanh Tung, Vu Thi Hien, Vu Quo Luan, Nguyen Phu Huy, Nguyen Hoang Lo, Duong Tan Nhut (217). Effet of metal nanopartiles on the growth of Ngo Linh ginseng (Panax vietnamensis) lateral roots ultured in vitro. Hue University Journal of Siene: Natural Siene, 126(1C): Nguyen Thi Nhat Linh, Nguyen Hoang Lo, Duong Tan Nhut (217). Effet of maronutrient medium, different arbohydrate soures and peptone on formation and development of seondary root in Panax vietnamensis adventitious root ulture. Journal of Siene and Tehnology, Hue University of Sienes University - Hue University, 1(1): Nguyen Thi Nhat Linh, Hoang Thanh Tung, Nguyen Hoang Lo, Duong Tan Nhut (217). The effet of bioti and abioti eliitors on biomass and saponin prodution of seondary root ultivated in shake flasks Panax vietnamensis adventitious root. Journal of Biotehnogy, 15(2): Nguyen Thi Nhat Linh, Hoang Thanh Tung, Nguyen Hoang Lo, Duong Tan Nhut (215). Enhaning seondary root formation and development from in vitro adventitious root of Ngo Linh ginseng (Panax vietnamensis). Journal of Biotehnogy, 13(2): Duong Tan Nhut, Tran Hieu, Nguyen Thi Nhat Linh, Hoang Thanh Tung, Nguyen Ba Nam, Nguyen Phu Huy, Vu Quo Luan, Vu Thi Hien (215). Optimum onditions for proliferation and saponin aumulation of Panax vietnamensis Ha et Grushv. adventitious roots in ulture systems. Journal of Biotehnogy, 13(3): Ho Thanh Tâm, Nguyen Ba Nam, Hoang Xuan Chien, Le Kim Cuong, Ngo Thanh Tai, Nguyen Viet Cuong, Nguyen Phu Huy, Trinh Thi Huong, Tran Hieu, Nguyen Thi Nhat Linh, Duong Tan Nhut (215). Optimization of ulture onditions and medium omposition for adventitious root indution from leaves of Panax vietnamensis Ha et Grushv. Journal of Biotehnogy, 13(3):