Tom tat tieng anh

and investigate elicitor on saporin accumulation of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.)” was studied to not only improve the formation and growth of lateral ro[r]

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 - 2017

Instructor:

Prof.Dr Duong Tan Nhut

Prof.Dr Nguyen Hoang Loc

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 can be found at:

- National Library of Vietnam;

- Learning Resource center – Hue University;

- Library Hue University of Sciences – Hue University

INTRODUCTION

Vietnamese ginseng, Ngoc Linh Ginseng (Panax vietnamensis

Ha et Grushv.) is an endemic ginseng special of Vietnam ginseng

This ginseng has been studied and showed many pharmaceutical ingridients than other specials of same genus, including 26 saponins like American ginseng and Korean ginseng; besides, there was found more than 20 other saponins in Ngoc Linh ginseng

These saponins help health improvement, vitality enhancement, blood pressure controll, antioxidants, cancer preventation, anti- aging, immune stimulation, treatment of malaria, depression, liver,

kidney and diabetes This valuable ginseng has been very scarce and ranked in the red list book that needs to be preserved, but it still over-exploited in nature Moreover, using compounds of Ngoc Linh ginseng in pharmaceutical industry was still difficulty

In vitro root culture is considered as potential alternative

method to havest a large biomass in a shortime In vitro Ngoc

Linh ginseng root was optimally grown when it was culture in the pertinent medium and conditions Although the increase of root biomass is important, the saponin accumulation is more nesessary For the reason that the largest root biomass having the highest saponin could be used as a raw material for the production of functional foods, even to produce drugs In fact, culturing Ngoc

Linh ginseng by in vitro roots, the root yeild developed quickly,

but saponin content is lower than the wild ginseng roots Elicitors have the great potential in enhancing the saponin accumulation in Ngoc Linh ginseng culture, so the research on the application of elicitors into the ginseng lateral root culture is very necessary

Therefore, the thesis “Improve secondary root culture of

and investigate elicitor on saporin accumulation of Ngoc Linh

ginseng (Panax vietnamensis Ha et Grushv.)” was studied to

not only improve the formation and growth of lateral roots (LRs)

from in vitro adventitious roots (ARs) by optimizing culture

medium and culture conditions, but also enhancing the accumulation of three saponin (MR2, Rb1 and Rg1) in Ngoc Linh ginseng via the effectiveness of elicitors

Objectives of thesis: Optimazize the in vitro Ngoc Linh ginseng

lateral root culture and multiplicate the root biomass is more stable and higher in bioreactor system Determine and evalute the effect

of different types, and concentrations of five elicitors (SA, JA, ABA, CHN và YE) on producing saponin compounds Next, combine in various types of elicitors to enhance effectiveness of the saponin accumulation, and determine the best elicitation time to

develop the highest saponin in LRs of Ngoc Linh từ ARs in vitro

Siencific significance: Providing sciencific valuable data about

root morphology, multiplication of root biomass and enhance

saponin content in culturing in vitro LRs of Ngoc Linh ginseng

In addition, the thesis is also a useful reference for the study and teaching in metabolism field, plant physiology and tissue culture

Practical significance: This study had established the culture

medium and culture condition for the development of in vitro Ngoc

Linh ginseng LRs culture in short time In addition, this study had determined the best of type, concentration and elicitation time for accumulating saponin compounds (MR2, Rg1 và Rb1) in LRs The results suggest that the treatment is optimized for increasing the growth and saponin content of LRs; thus, the ginseng saponin production could be scale-up via root culture

CHAPTER 1 LITERATURE REVIEW

NGOC LINH GINSENG

1.

Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) was

found at Ngoc Linh mountain, DacTo district, Kon Tum province This ginseng has been studied and showed that it has many

pharmaceutical compounds than other Panax genus

ADVENTITIOUS ROOT (AR) and LATERAL ROOT (LR) 2.

CULTURE

LRs originate from pericycle cells of existing roots like the PR, earlier LRs, or ARs Depending on the species, they may derive from pericycle cells adjacent to xylem pole or to phloem pole cells of dicot plant suchs as Ngoc Linh gingseng In monocot plant, the endodermal cells also contribute to LR development and produce the epidermis and the root cap, whereas the pericycle cells are the source

of all remaining tissues The different stages of LR initiation and development have been tarting from the first cell division in the pericycle cell up to the emerging LR primordium, seven stages have been identified that correspond to different steps in the acquisition of cell identity and tissue organization In contrast to LRs, ARs develop from different tissues and consequently from different cell types A conserved feature is that ARs always develop from cells neighboring vascular tissues, whether they are part of the plant’s development program or are artificially induced by wounding or hormone applications Often, cell division had already started and/or small AR primordia were already formed

SAPONIN COMPOUNDS

3.

Saponins are naturally occurring surface-active glycosides It is a diverse group of terpenoids characterized by their structure Saponins are composed of a steroidal or triterpenoid aglycon and one or more sugar chains A plant might contain several dozen saponins with closely related structures The latest research of pharmacological compounds showed that saponins of Ngoc Linh ginseng have over 60 types; the most important group of the ginseng secondary metabolites is the saponin triterpene which the main representatives are MR2, Rb1 and Rg1 in the dammaran group Triterpene saponins are complex structures, so the saponin synthesis is not economically feasible for producing on a large scale, and the wild ginseng sources

are extremely rare

ELICITOR and ELICITATION

4.

The term “elicitor” is very general and refers to chemicals from various sources, biotic or abiotic, as well as physical factors, that can trigger a response in living organisms resulting in accumulation of secondary metabolites Elicitation is the induced or enhanced biosynthesis of metabolites due to addition of trace amounts of

elicitors So far, studies on the application of elicitors to in vitro

culture of Ngoc Linh ginseng to improve the content of saponins are

very limited Duong Tan Nhut et al., (2012) initially assessed the

effect of MeJA on the saponin accumulation in the ginseng calli Trinh Thi Huong (2017) has also begun to study the effects of MeJA,

SA and ABA to enhance saponin compounds in the transgenic root culture

CHAPTER 2 MATERIALS AND METHODS

1 ROOT EXPLANTS

In vitro adventitious root clusters of P vietnamensis, which were

induced from leaves (1×1 cm) and grown for 40 days on SH medium (Schenk and Hildebrandt 1972) supplemented with 5 mg/L IBA, 30 g/L sucrose, and 8 g/L agar, were used as the source of explants

2 RESEARCH CONTENTS

2.1 Optimal explant resource and medium culture for Ngoc Linh

ginseng LRs growth from in vitro ARs

2.1.1 Investigation of the effect of the in vitro ARs cuttings on the

formation and growth of Ngoc Linh ginseng LRs

2.1.2 Investigation of the effect of auxin on the formation and

growth of Ngoc Linh ginseng from in vitro ARs

2.1.3 Investigating the effect of the combination of auxin and cytokinin on the formation and growth of Ngoc Linh ginseng LRs 2.1.4 Investigation of the influence of mineral medium on the formation and growth of Ngoc Linh ginseng LRs

2.1.5 Investigating the effects of NH4

+

/NO3 -

in MS medium on the formation and growth of Ngoc Linh ginseng LRs

2.1.6 Investigation of the effects of carbohydrates on the formation and growth of Ngoc Linh ginseng LRs

2.1.7 Investigation of the effect of culture medium on the formation and growth of Ngoc Linh ginseng LRs

2.2 Optimized uncultured root culture conditions of Ngoc Linh

ginseng to enhance in vitro LR formation and saponin accumulation

2.2.1 Investigation of the effect of culture time on the growth and accumulation of saponins of Ngoc Linh LR ginseng

2.2.2 Investigation of the effect of culture temperature on the growth

and accumulation of saponins of Ngoc Linh ginseng LRs

2.2.3 Investigation of the effect of pH on saponin growth and

accumulation of Ngoc Linh ginseng LR from in vitro ARs

2.2.4 Investigation of the effect of dark and light culture on the growth of Ngoc Linh ginseng LRs

2.2.5 Investigation of the effect of culture volume on the formation

and growth of Ngoc Linh ginseng LR from cultured in vitro ARs

2.2.6 Investigation of the effect of culture system on the growth and accumulation of saponins of Ngoc Linh ginseng LRs

2.3 Optimal saponin accumulation of LRs formed from the ARs of Ngoc Linh ginseng via the effect of elicitors

2.3.1 Investigation of the effects of chitosan on the growth and

saponin accumulation of Ngoc Linh ginseng LR from in vitro ARs

2.3.2 Investigation of the effect of yeast extract on the growth and saponin accumulation of Ngoc Linh ginseng LRs

2.3.3 The effect of abscisic acid on the growth and saponin accumulation of Ngoc Linh ginseng LRs

2.3.4 Investigation of the effect of salicylic acid on the growth and saponin accumulation of Ngoc Linh ginseng LRs

2.3.5 Investigation of the effects of jasmonic acid on the growth and saponins accumulation of Ngoc Linh ginseng LRs

2.3.6 Investigation on the combined effect of exogenous elicitor and endogenous elicitor on the growth and saponin accumulation of

Ngoc Linh LR in vitro

2.3.7 Investigation on the effect of elicitation time on the growth and

saponin accumulation of in vitro Ngoc Linh LR

2.4 Determination of growth rate, dry matter percentage, LR formation rate and proliferation of the culture

2.6 Culture conditions

The in vitro Ngoc Linh AR cultures were maintained in the darkness

cabinet with temperature 25 ± 2°C and relative humidity 55 - 60% Except for solid culture, the root culture was shaken on the Innova

2100 plantform shaker (Hermle, Germany) at 100 rpm

3 SAPONIN ANALYSIS

3.1 Thin layer chromatography on the saponin analysis

3.2 High performance liquid chromatography

3.4 Determination method of saponin productivity of ginseng LRs

4 CYTOLOGY METHODS

5 QUANTITATIVE DATA ANALYSIS USING SPSS 20.0

CHAPTER 3: RESULTS

1 OPTIMIZE NGOC LINH GINSENG LR CULTURE FROM

IN VITRO ARS

1.1 Optimal explant resource and medium culture for Ngoc Linh

ginseng LRs growth from in vitro ARs

1.1.1 Effect of the in vitro ARs cuttings on the formation and growth of Ngoc Linh ginseng LRs

The results of adventitious root (ARs) influent study on the

formation and growth of in vitro Ngoc Linh ginseng the lateral root

(LR) showed in Table 3.1 The LR source from the petiole pattern has the size of 2 cm, no separate the pattern is the most suitable for

the formation and growth of in vitro Ngoc Linh ginseng LRs

Table 3.1 Effect of the in vitro ARs cuttings on the formation and

growth of Ngoc Linh ginseng LRs

LR formation rate (%)

LR number

LR lenght (cm)

FW (mg)

1.1.2 Effect of auxin on the formation and growth of Ngoc Linh ginseng from in vitro ARs

Table 3.2 Effect of auxin on the formation and growth of Ngoc Linh

ginseng from in vitro ARs

Auxin

(mg/L)

LR formation

rate (%)

LR number

LR lenght (cm)

FW (mg)

DW (mg)

Table 3.3 Effect of the combination of IBA and cytokinin on the

formation and growth of Ngoc Linh ginseng LRs

LR number

LR length (cm)

FW (mg)

DW (mg)

7,0

0 94.53 a * 13.75 a 5.25 a 155.00 de 14.25 de

Kin

0.1 81.53b 5.75c 0.95de 85.50g 6.50hi0.5 68.75 cd 4.00 de 0.75 ef 121.00 f 13.75 def

1.1.4 Effect of mineral medium on the formation and growth of Ngoc Linh ginseng LRs

Table 3.4 Effect of mineral medium on the formation and growth of

Ngoc Linh ginseng LRs

LR length (cm)

FW (mg)

DW

SH 78.7b* 16.5b 3.63b 206.8c 20.8b LRs and ARs in white and slender

MS 92.5 a 18.3 a 4.50 a 346.3 a 31.3 a Yellowing thick ARs Big LRs and

B5 82.5ab 14.0c 1.35c 312.3b 21.8b

Yellowing or necrosis LRs and ARs

* Different letters indicate significant difference (P < 0.05) with Duncan’s test

1.1.5 Effect of NH 4

+

/NO 3 -

rate in MS medium on the formation and growth of Ngoc Linh ginseng LRs

Table 3.5 Effect of NH4

+

/NO3 -

rate in MS medium on the formation and growth of Ngoc Linh ginseng LRs

FW (mg)

DW (mg) 0/2 15.22c* 4.33bc 323.67b 24.33b

Graph 3.1 Comparisons between the modified medium had the best NH4 /NO 3

-ratio compared to other culture media

* Different letters on each column indicate significant difference (P < 0.05) with

Duncan’s test FW: Fresh weight, DW: Dry weight

1.1.6 Effects of carbohydrates on the formation and growth of Ngoc Linh ginseng LRs

Table 3.6 Effects of carbohydrates on the formation and growth of

Ngoc Linh ginseng LRs

Carbohydrate

(g/L)

FW (mg)

DW (mg)

Dry matter rate (%)

LRs and ARs

0 51.67 e * 3.33 hi 6.42 d Necrosis

D-Glucose

10 160.33 d 15.00 gh 9.82 bc white, long and slightly branch

30 206.67 d 18.33 g 8.89 bcd White, very long, slightly branch

40 379.33 c 41.57 e 10.96 ab White, very long, big, less branch

50 577.67b 51.93bcd 8.99bcd White, very long, big, less branch

60 523.00 b 67.40 a 13.03 a White, very long, big, less branch

Sucrose

10 374.33 c 29.33 f 8.85 bcd White, very long, big, less branch

40 572.33 b 56.00 b 9.79 ab White, very long, big, high branch

50 533.33 b 47.83 cde 9.16 bcd White, very long, big, high branch

60 526.67 b 46.33 de 7.80 cd White, very long, big, high branch

Maltose

10 91.67 e 9.00hi 8.22 bcd Yellow, less branched or prolonged

40 181.67 d 17.33 f 9.76 bc Yellow and necrosis, less branched

50 102.73 e 7.67 hi 7.37 cd Yellow and necrosis, less branched

60 62.53e 4.33hi 6.94cd Yellow and necrosis, less branched

*Different letters indicate significant difference (P < 0.05) with Duncan’s test