SOMATIC EMBRYO LNDUCTION AND PLANT RECENERATION iN AMERICAN

GINSENG (Panax quinquefdium L.)

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

XIAOLAN WANG

In partial fulfilment of requirements

for the degree of

Master of Science

December, 1997

O Xiaolan Wang

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ABSTRACT

SOMATIC EMBRYO INDUCTION AND PLANT REGENERATION IN

AMERICAN GINSENG (Panax quinquefolium L.)

Xiao lan Wang University of Guelph. 1 997

Advisor: Professor John T.A. Proctor

Plant regeneration of American ginseng through somatic embryogenesis was obtained in four

months. Somatic embryos (SES) were induced on auxin supplemented MS medium from three types

of explants and up to 68 % of the explants produced SES afier 8 weeks of culture. Embryogenesis

was affected by a w i n type, concentration, and explant type. Generally, the frequency of

embryogenesis increased with NAA. and reduced with 2,443 or dicamba as the concentration of

auxins changed from 5 to 15 FM. Including cytokinin, 0.1 to 5 FM BAP or kinetin. in auxin

containing medium generaily inhibited auxin-induced somatic embryogenesis; the influence was

also dependent on the explant type.

Over 80 % of the SES converted into plantlets on half strength MS medium supplemented

with 1.45 FM GA3 d e r 2 weeks of culture in the light. In vitro flowering was observed in plantlets

obtained fron SES afier 10 weeks of culture. Regenerated plantlets contained the sarne six major

ginsenosides as those found in the roots of field grown plants.

ACKNOWLEDGEMENTS

1 would like to express my sincere thanks to my advisor Dr. John T. A. Proctor, for his

guidance, encouragement, patience and constant support. 1 would also like to thank Drs.

Praveen K. Saxena and Rick D. Reeleder, Agriculture and Agri-Food Canada, for their

advice regarding my research project and cntical evaluation of this manuscript.

Special thanks goes to Jill Shupe for her assistance with embryo culture. To Dr.

Yukio Kakuda, Food Science, for his proficiency in HPLC assay. Also to Sandy Smith. Food

Science, and Lewis Melville. Botany, for their help with scanning electron microscopy; to

Helga Hunter, OVC histology laboratory, for her expertise in embedding of the sarnples.

Thanks go to Dr. Williams Matthes-Sears, Statistics service, for his suggestions and

guidance with statistical analyses. Thanks Dn. Hai Yu and Pinggao Zhang for their help, and

to Dr. Sankaran Krishnaraj for his mitical reading of this manuscript.

I am grateful to Dean Louait, Tannis Simmon, Murthy Srivinvasa, Jemn Victor, Paul

Banks and Susan Murch for their help in various aspects of this research project. To my

fellow graduate students, th& you for your help. Thanks to the Chinese students and fnends

in Bovey Building for their support and fiiendship.

To my husband Jirong: 1 express my sincere gratitude for your encouragement and

suggestions. Also to my parents for their constant support. To my son Alan for his

understanding and patience.

This research was supported by the N a d Sciences and Engineering Research

Council of Canada and Agriculture and Agi-Food Canada Research Partnership Program

with Gerald Nelson Farrns Ltd. and J. C. K. Farms Ltd.

TABLE OF CONTENTS

ABSTRACT

ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

. . TmLEOFCONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i l

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LISTOFTABLES iv

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LISTOFFIGURES vi

... LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

. CHAPTER ONE Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1Introduction i

1.2 Ginsenosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.3 Somatic embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.4 Sornatic embryogenesis in ginseng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

CHAPTER TWO . Induction of somatic embryos . Effects of pretreatment. sucrose concentrations. auxin type and concentration on different types of explants of Americanginseng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1Introduction 36 3.2 Materiais and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 3.4Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary 49

CHAPTER THREE . Induction of somatic embryos . Effect of cytokinin in auxin supplemented medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 7 1 3.1Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 3.2 Materids and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 7 4 3.3Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 3.4Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary 83

CHAPTER FOUR . Developmetit of Plantlets obtained fkom somatic embryos of Amencan ginseng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 8 8 4.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 9 1

4.3Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4Discussion 99

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary 103

CHAPTER FIVE . Ginsenoside content of regenerated plantlets of American ginseng 122 5.lIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Materials and methods 124 5.3Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4Discussion 130 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

. . . . . . . . . . . . . . . . . . . . . . . . . . . GENERAL DISCUSSION AND CONCLUSIONS 134

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

LIST OF TABLES

Table 1.1 Ginsenoside content of callus and regenerated plants from somatic embryos of Orientai ginseng ( P a n a ginseng C. A. Meyer) . . . . . . . . . . . . . . . . . . . . . . . . . - 2 0

Table 1.2 Ginsenoside content of original plants and tissue culture materials of Oriental ginseng (Panax ginseng C. A. Meyer) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1

Table 1.3 Somatic embryogenesis in Oriental ginseng ( P a n a ginseng C. A. Meyer) . 22

Table 1.4 Plant regeneration frorn somatic embryos in Oriental ginseng (Panax ginseng)

Table 1.5 Somatic embryogenesis in American Ginseng (Panax quinquefolium L.) . . .24

Table 1.6 Plant regeneration fiom somatic embryos in Amencan Ginseng (Panax quinquefolium L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 2 5

Table 2.1 Effect of 2,4-D, NAA, and dicamba on somatic embryogenesis in cotyledonary and zygotic embryo explants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1

Table 2.2 Effect of 2,4-D, NAA, and dicamba on somatic embryogenesis in shoot expiant ofAmericanginseng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Table 2.3 Main effect of 2,4-D and NAA on sornatic embryogenesis in cotyledonary, seedling segments, zygotic embryo, and shoot explants . . . . . . . . . . . . . . . . . . -53

Table 2.4 Distribution of somatic embryos induced from seedling segments on 3 concentrations of 2,4-D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Table 2.5 Effect of exposure time to 5 FM 2,4-D on somatic embryogenesis in cotyledonary, zygotic embryo, and shoot explants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5

Table 2.6 Main effect of light and GAj as pre-treatment on growth of seedlings from stratified and green seeds on subsequent somatic embryogenesis in cotyledonary explants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Table 3.1 The effect of cytokinins, kinetin and BAP, supplemented to 2,443 containing medium on somatic embryogenesis in cotyledonary and shoot explants . . . . . . 84

Table 3.2 The effect of cytokinin in NAA containing medium on somatic embryogenesis inshootexplants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Table 3.3 The effect of two cytokinins, kinetin and BAP, in dicamba containing medium on somatic embryogenesis in cotyledonary and shoot explants . . . . . . . . . . . . . . . 8 6

Table 4.1 Effect of GA, and BAP on rates of plantlet, shoot and other SES ....... 104

Table 4.2 Effect of pretreatment for seedling growth on rates of plantlet, shoot and other . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SES. 105

Table 4.3 Effect of GA, and BAP on rates of plantlet shoot and other SES and growth of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . plantlets 106

Table 4.4 Effect of sucrose and casein hydrolysate (CH), and sucrose and ABA on growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . of plantlets 107

Table 4.5 Cornparison of germination of seeds, zygotic embryos and somatic embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

LIST OF FIGURES

Figure 2.1 Seed. zygotic embryo. and seedlings of Amencan ginseng . . . . . . . . . . . . . . 57

Figure 2.2 Cotyledonary explants cultured on 5 pM 2.4-D and 15 pM NAA supplemented medium for 2 weeks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Figure 2.3 Cotyledonary explants responded differently with different auxins . . . . . . . . 59

Figure 2.4 Zygotic embryo explants after 5 weeks of induction . . . . . . . . . . . . . . . . . . . 60

Figure 2.5 Shoot explants after 8 weeks of induction . . . . . . . . . . . . . . . . . . . . . . . . . . . - 6 1

Figure 2.6 Effect of a combination of 2. 4.D and NAA on somatic embryo (SE) induction with cotyledonary (A. B) and zygotic embryo ( C) explants . . . . . . . . . . . . . . . . 62

Figure 2.7 Effect of a combination of 2. 4.D and NAA on somatic embryo (SE) induction with shoot explants afler 7 weeks of induction . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Figure 2.8 Somatic embryo (SE) induction in coty ledonary explants cultured on 5 pM 2. 4.D supplemented MS medium for various times . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Figure 2.9 Somatic embryo (SE) induction in shoot explants cultured on 5 pM 2.4-D enriched medium for various times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -65

Figure 2.1 0 Coty ledonary explants cultured on 5 pM 2. 4.D and 1 5 FM NAA supplemented medium for 8 weeks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Figure 2.1 1 Somatic embryogenesis in green seeds with cotyledonary explants on 2.4-D (5 pM) and NAA (15 pM) supplemented MS medium . . . . . . . . . . . . . . . . . . . . . . 67

Figure 2.12 Responses of zygotic embryo (ZE; A. B) and shoot (C. D) explants to MS basal media without growth regdators but containing different levels of sucrose for

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . differenttimes 68

Figure 2.13 Histology snidy of somatic embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . -69

Figure 2.14 SEM observation of somatic embryogenesis after 8 weeks of induction on 2. 4.D containing medium (A-D) and a zygotic embryo (ZE; E) . . . . . . . . . . . . . 70

Figure 3.1 Effect of cytokuiin in 2. 4.D containing MS medium on somatic embryogenesis with cotyledonary explants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Figure 4.1 In vitro regeneration of American ginseng . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Figure 4.2 Plantlet with two stems converted fiom a somatic embryo obtained from green . . . . . . seed. after 3 weeks culture on 1.45 pM GA, containhg % MS medium 110

. . . . . . . . Figure 4.3 Plantlets converted fiorn somatic embryos after 4 weeks culture I l 1

Figure 4.4 Flowers of greenhouse-grown 3-year-old plants . . . . . . . . . . . . . . . . . . . . . . 112

. . . . . Figure 4.5 An inflorescence developed fkom a somatic embryo-derived plantlet 113

Figure 4.6 An inflorescence observed in an somatic embryo-derived plantlet. having 8 flowers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i l 4

Figure 4.7 Flowers of somatic embryo-derived plantlets . . . . . . . . . . . . . . . . . . . . . . . . 115

Figure 4.8 Anther and pollen grains of somatic embryo-derived plantlets . . . . . . . . . . . 116

Figure 4.9 SEM micrograph of anthers. pollen grains. and surface of pollens of greenhouse grown 3-year-old plants and somatic embryo derived plantlets . . . . . . . 117

Figure 4.10 Plantlets developed from somatic embryos . . . . . . . . . . . . . . . . . . . . . . . . . 118

Figure 4.1 1 Three-month-old plantlet developed fiorn a somatic embryo . showing three well developed shoots . a tap root and lateral roots . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Figure 4.12 Roots of greenhouse grown plants (a) and somatic embryo derived plantlets (b) after 3 months storage at 3 O C . showing shoot emergence from the peremating bud .

Figure

Figure

4.13 Germination of ernbryos afker 9 days culture . . . . . . . . . . . . . . . . . . . . . . . 121

5.1 Distribution of ginsenoside content and Rb and Rg groups and ratios invarious samples of American ginseng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

ABA

BAP

B5

CH

Dicamba

g

GA,

MS

NAA

PM

2,4-D

LIST OF ABBREVIATIONS

Abscisic acid

Benzy laminopurine

Gamborg et al., ( 1 968) medium

Casein hydrolysate

3,6-dichioro-O-anisic acid

Gram

Gibberellic acid

Murashige and Skoog ( 1962) medium

Naphthaleneacetic acid

Micro molar

2,4-dichlorophenoxyacetic acid

Chapter 1. Literature Review

1.1 Introduction

Ginseng is one of the world's most valued plants, and has been cailed green gold (Persans,

1986). It is an econornically important crop in international trade for its highly valued root.

Chinese have used ginseng for thousands of years as a wonder cimg, cure-dl, and

aphrodisiac (Proctor and Bailey, 1987). In Li Shih-Chen's Pen-ts'aoKang-mu (Matenal

medica with commentaries, 1596), ginseng was described as a superior article arnong

traditional Chinese herbals (Hu, 1976). It is now believed that the pharmacological properties

of ginseng is because of its active ingredient saponins, also commonly known as

ginsenosides (Homok, 1992). Ginseng has been used for its effects as an anti-stress, enhancer

of blood circulation, nerve growth and as an anti-inflarnrnatory substance (Hikino, 199 1).

Ginseng is a dicotyledonous plant in the genus of Pana, farnily Araliaceae. Two

species of Panax are of commercial importance: Oriental ginseng (Panax ginseng C. A.

Meyer) which is native to northeastem China and Korea, and Amencan ginseng ( P a n a

qztinquefolium L.) which is native to parts of eastem Canada and the U.S.A., fiom Quebec

to Louisiana, Alabama and Arkansas (Oliver, 1992). In Canada, Pana quinquefolium was

fint discovered in 171 6 by a Jesuit missionary, Father Lafitau (Garman, 1898), and people

began collecting it for export to China shortly afterwards. By the late 1 800ts, people started

making attempts to grow Arnerican ginseng in a small way (Garman, 1898).

Over 80% of the ginseng grown in North America is exported to Hong Kong which

is the key player in the international trade of ginseng (Fisher, 1993; But et al., 1995). The

ginseng industry in North Amenca is expanding rapidly with an estimated 1 140 t, worth $

55 million. harvested in Ontario in 1996 (Proctor. unpublished data).

1.1.1 The plant

American ginseng is a perennial herbaceous plant with a soiitary stem, whorled leaves, and

a thick taproot ( Lewis et al., 1982; Proctor and Bailey, 1987). Ginseng flowers are small and

pentamerous (Lewis and Zenger. 1982). Arnerican ginseng grows naturally under the canopy

of hardwood forests. It is a shade-loving plant (Hu et al., 1980). The aboveground portion

of the plant dies at the end of each growing season, and the new stem develops in the

following spnng fkom the bud on the rhizome (Hu et al., 1980). The root continues to grow

each growing season.

1.1.2 Cultivation and Propagation

As a shade-loving herb, Arnerican ginseng is grown on raised beds, with a protective mulch

of straw or other material, and under natural or artificial shade (Proctor and Bailey. 1987).

it requires 3-4 yean of growth before harvest (Fisher, 1993; Oliver et al., 1992). Another

lirniting factor in ginseng production is disease caused by fungal pathogens. The yield of

ginseng can be reduced by 60-80% because of damping off, root rot, Alternaria blight and

other diseases (Proctor and Bailey, 1987; Proctor 1996).

Seeding is the principal rnethod of propagating ginseng (Proctor and Bailey, 1987).

A typical flowenng, 4-year-old American ginseng plant may cary 30-40 bemes in each

inflorescence, and each beny has an average of two seeds, which are 5-6 mm long and 4-5

mm wide (Proctor and Bailey, 1987). Seeds are usually harvested fiom 3- to 4-year-old

plants in August / Septernber when the bemes are crimson red, dthough smail arnount of

seeds can be produced by 2-year-old plants (Oliver et al., 1992). Typically. 150-250 pounds

of seeds/acre can be harvested (Fisher, 1993). The embryo of ginseng seeds at harvest is

immature. Stoltz et al., (1985) reported that American ginseng has an immature embryo

which is 0.4 to 0.5 mm long at the time the h i t ripens. A cool-wam-cool temperature

treatment over a 18- 22 months stratification is generally required for embryo development

and seed germination (Proctor and Louttit, 1995). Comrnonly, seeds are stratifîed outdoors

in boxes mixed with moist sand for 12 to 14 months and planted in the field in the fall.

Germination occurs the following spnng.

It has been demonstrated recently that the effect of a combination of seed treatment

temperature regimes and growth regdators, particularly gibberellic acid on early stratification

for "green" (i.e., August / September harvested) seeds allows them to germinate in the

following May (Hovius, 1996).

1.2 Ginsenosides

Ginseng saponins, or ginsenosides, are the pharmacologically active constituents of ginseng

(Inomato et al., 1 993; Yoshimatsu et al., 1996). Several pharmacological properties have

been reported for ginsenosides including effects on the centrai nervous system, protection

from stress ulcers, antifatigue action. endocnnological effects, and acceleration of

metabolism of carbohydrates, lipids, and proteins (Li et al., 1996).

More dian 30 ginsenosides have been isolated from ginseng plants , of which Rb,,

Rb?, Rc, Rd. Re, and Rg, are the six major ones (Li et al., 1996; Smith et al., 1996). These

can be divided into two main groups: Rb group (including Rb,, Rb,, Rc, and Rd) having

protopanaxadi01 as the sapogenin, and the Rg group (including Re and Rg,) having

protopanaxatriol (Yoshikawa and Funiya 1987). The ratio of the Rb / Rg groups is ofien

used as an indicator in qualitative evaluation of ginseng (Asaka et al., 1 994b; Yoshikawa and

Furuya, 1987). High-performance liquid chromatography (HPLC) has often been used for

the analysis of ginsenosides in ginseng extracts and products (Soldati and Sticher, 1980).

1.2.1 Localization of ginsenosides

In 6-year-old Oriental ginseng, ginsenosides were found outside the cambium in the root

tissue, particularly in the periderm and outer cortex, and only traces of ginsenosides were

found in the xylem and pith (Kubo et al., 1 980; Tani et al ., 1 98 1). In Amencan ginseng,

heavier roots contained more ginsenosides on a weight basis, and the three fractions: root

fibre; periderm and cortical tissue; xylem and pith tissue. had similar amounts of

ginsenosides (Smith et al., 1996).

1.2.2 Variation in ginsenoside contents

Ginsenoside contents are influenced by many factors such as plant species. age of plants.

growing conditions and soi1 fertility (Lewis, 1982: Li et al., 1996; Konsler et al., 1990;

Soldati and Sticher. 1980).

Quantitative differences in total and individuai ginsenosides between American and

Oriental ginseng and wild and cultivated American ginseng were shown by Betz et al.,

(1 984). Konsler et al. (1 990) observed that leafginsenoside content was positively correlated

with the soi1 fertility. In Oriental ginseng, the total yield of ginsenosides per plant increased

significantly with age from one to six yean (Soldati and Tanaka, 1984). Kim et al., (198 1)

found seasonal variations in both Rb and Rg groups of ginsenosides in roots of Oriental

ginseng were significantly higher in summer than in winter.

Large variations in the dry weight and ginsenosides content of Amencan ginseng

roots have also k e n reported (Smith et al., 1996). Among twenty 4-year-old roots harvested

from a homogeneous-looking lm-' plot, the dry weight of the roots ranged from 3.5 to 22.8

g, and the ginsenoside levels of the roots varied from 24.7 to 56.4 pmol g". Smith et al.,

(1996) suggested that genetic diversity was partly the cause of the variation, since the

environmental variation was minimal.

1.2.3 Production of ginsenosides in tissue culture

Production of ginsenosides in ginseng tissue culture using selected ce11 lines has been

reported by several authors. The production has been generally found to be affected by

various factors including culture methods and age (Mathur et al.. 1994), medium

composition (Choi et al., 1994; Funiya et al., 1983; Odnervail and Bjork, 1986a b;

Yoshikawa and Fumya, 1987). and culture conditions (Choi et al., 1994; Funiya et al.. 1983).

Asaka et al., (1994b) compared the ginsenoside content of seedlings with plantlets

regenerated fiom sornatic ernbryos of Oriental ginseng. Seedlings used were 2-3 months old

and 6- 10 cm tall, whereas plantlets were those regenerated fiom multiple shoots-derived SES.

Ginsenoside levels in callus, somatic embryos, shoots, and roots of both seedlings and

plantlets were determined by HPLC (Table 1.1). Although regenerated plantlets and

seedlings were similar in terms of biosynthesis, ginsenoside content in callus was lower than

somatic embryos or plantlets, indicating that ginsenoside production was induced by tissue

differentiation.

Furuya et al., (1986) reported ginsenoside production in callus, multiple shoots,

adventitious root (in callus or suspension culture). and original plants (Table 1.2). They

found that the multiple shoots and adventitious roots produced larger amounts of

ginsenoside than the original callus.

1.3 Somatic em bryogenesis

Hanning (1904) observed that the nearly mature embryos of Raphanm, when grown on

mineral salts. sugars, amino acids and plant extracts. produced transplantable seedlings.

Since then, zygotic embryo culture has been used to break seed dormancy, to shorten

breeding cycle times. to study basic processes in embryogenesis. to rescue immature hybrid

embryos and to provide microcloning source material (Hu and Wang. 1986).

"AI1 somatic cells within a plant contain the entire set of information necessary to

create a complete and hct ional plant" (Merkle et al.. 1995). A somatic embryo (SE) is a

bipolar structure that develops fiom somatic cells and is capable of developing into a whole

plant in a manner analogous to a seed embryo. Somatic embryogenesis was first reported in

carrot (Reinert, 1958; Steward et al., 1958), and has been demonstrated in numerous species

(Brown et al.. 1995: Dunstan et al., 1995; Kx-ishnaRaj et al.. 1995). Most somatic embryos

(SES) pass through the same stages of development as a zygotic embryo. i.e.. globular. heart-

shaped, torpedo- and cotyledonary stage. In addition. most SES contain embryo specific

macro-molecules like storage proteins and lipids.

Somatic embryos have been induced fiom a variety of plant tissues, most frequently

from zygotic embryos, germinating seedlings, shoot meristem, and young flowers or

inflorescences (Merkle et al., 1995). Because of their high regenerative potential, immature

6

zygotic embryos have often been used for in vitro clonal propagation and are excellent

explants. h o s t al1 SES in conifers and deciduous trees are derived from zygotic ernbryos

(Deunff, 1995).

The process of regeneration involves, in sequence, the initiation of a bipolar structure,

development and maturation of SES, conversion to plantlets, and transfer of plants out of

culture medium into soil or soil mixture. At each of these stages, loses and limitations reduce

the potential number of regenerated plants (Merkle et al.. 1995). Somatic embryogenesis is

a two-step-process in sorne cases (Molle et al., 1993; McKersie and Bowley, 1993):

involving first, induction of proembryogenic masses in a medium containing exogenous

auxin. and then regeneration of SES in a low auxin or awcin-fiee medium. The quantity and

type of growth regulaton in the SE inducing medium is one of the major factors influencing

regeneration in alfalfa (McKersie and Bowley, 1993). Auxins, especially 2.4-

dichlorophenoxyacetic acid (2,4-D) have been frequently used for SE induction. particularly

when intemenhg calli were involved. though it is not essential in some cases where SES can

be induced on basal medium without growth regulators. Pence et al.. (1 980) reported that SES

of Theobroma were produced directiy fiom the cotyledon in a medium without growth

regulators; however, higher fiequency of embryos were induced when an auxin (indole-3-

acetic acid, IAA or 2,4-D) and coconut milk were added to the medium. On the other hand,

2,443 combined with a cytokinin appeared more suitable for the proliferation of embryogenic

callus than 2,4-D alone in some cases such as creeping bentgrass (Zhong et al., 1991).

Cytokinins alone have also been w d as inducing agents for SES formation in some plants.

For example, thidianiron (TDZ), an active cytokinin-like substance, has been used for

stimulating somatic embryogenesis in many plants, especially in legumes and most dicot

species as well tree species (Neuman et al., 1993; Saxena et ai.. 1992). Genotype, tissue

type, and developmental stage of the donor plants may al1 be factors in determinhg the

response to auxin or cytokînin (Merkle et al., 1995).

1.4 Somâtic embryogenesis in ginseng

Early research on tissue culture of ginseng has been reviewed (Jhang et al., 1974; Chang and

Hsing, 198Oa; Proctor and Bailey, 1987). In 1964, Shi-Wei Luo reported that ginseng callus

was cultured for producing medicinal substances. Butenko(l968) first reported in vitro SE

induction of ginseng fiom root and embryo-derived callus. This literanire review will focus

on plant regeneration through somatic embryogenesis in the major ginseng species, Oriental

and American, although somatic embryogenesis has been obtained in P a n a notogenseng

recently from petiole and young flower buds on MS medium supplemented with 4.5 pM 2.4-

D (Shoyama et al., 1997).

1.4.1 Oriental ginseng

1.4.1.1 Callus induction and somatic embryogenesis (Table 1.31

Callus and somatic embryos (SES) have been induced fiom various sources of explants,

including root (Chang and Hsing. 1980a; Choi et al., 1982), stem and leaf (Cellirova et al.,

1992; Kishira et al., 1992). young flower bud (Kishira et al., 1992; Shoyarna et al., 1988),

zygotic embryo (Arya et al.. 1993: Choi et al., 1996; Ham and Lee, 1974; Lee et al., 1990),

cotyledon (Choi and Soh, 1996; Choi et al., 1984; Lee et al., 1993, and adventitious shoot

(Asaka et al., 1993; Asaka et al., 1994a). Jhang et al., (1974) established callus and

suspension cultures with explants obtained fiom one-year-old plants, and plantlets were

occasionaily observed with suspension culture.

Zygotic embryos or cotyledons have been used for SE induction on MS medium

supplemented with 2,4-D alone or combined with cytokinin. Lee et al., (1 990) demonstrated

the efEect of a combination of 2,4-D (0-45 PM) and 6-furfurylamino-purine (kinetin) (0-4.6

FM) on callus and SE fornation from mature zygotic embryos in darkness. Callus arose from

the penpheral region of the cotyledons on al1 media after 4 weeks of incubation. AAer 6

weeks callus gave nse to SES on the entire surface of the zygotic embryos, including

cotyledons. hypocotyls, and radicles. The most effective combination was 4.5 pM 2.4-D and

0.045 pM kinetin resdting in 55% fiequency of SE induction. Somatic embryos of different

development stages often coexisted on an individual explant, and many of the SES had

multiple cotyledons.

Arya et al.. (1993) also obtained SES from zygotic embryo explants on MS medium

supplernented with 4.5 pM 2.4-D and 0.046 FM kinetin. These SES were isolated from the

callus and individually cultured on MS basal medium alone. or supplemented with various

plant growth regdators to obtain adventitious embryogenesis. The cultures were incubated

under I6h-photoperiod at 20 OC. Auxins (2.4-D; naphthaleneacetic acid, N U : MA).

particularly 2.4-D, were effective for the induction of secondary and tertiary somatic

embryos. Cytokinins (kinetin and benzylaminopurine, BA) were not effective. In addition,

adventitious somatic embryogenesis occurred only on cotyledonary stage primary somatic

embryos, while globular and heart shaped SES always formed callus.

By using a two-step procedure in which MS medium containing 2,4-D (0.45 - 36.16

pM) was used followed by subculture ont0 1/2 strength MS medium supplemented with 2,4-

D and kinetin, SES were formed on cotyledonary explants in darkness (Choi et al., 1984).

For the first step of the culture, 22.6 FM 2,4-D was optimal and SES were induced in 5

months. Agrobacteriurn tumifaciem transformed coty ledonary explants have been used for

SE production on MS medium supplemented with 4.5 p M 2,4-D and 0.46 pM kinetin (Lee

et al., 1995).

Recently, direct SE induction has also been reported from intact zygotic embryos,

excised cotyledons, or cotyledon transverse thin ceIl layers (tTCL). Choi and Soh (1996)

reported that the developmental stage of zygotic embryos was critical for SE induction from

intact zygotic embryos on MS basal medium. As the embryos matured (from Imrn to 6 mm

long), the germination rate increased and SES formed only on ungerrninated immature

zygotic embryos. Mature zygotic embryos (4 mm long) did not form SES but most

germinated, whereas 76% of the immature zygotic embryos (2 mm long) induced somatic

embryos . However, at a very young stage (lmm long), zygotic embryos did not germinate

nor form somatic embryos. On the other hand, when mature zygotic embryos were cultured

on MS basal medium as isolated cotyledons, plumule, and radicle, only cotyledons produced

SES. Furthemore, SE production of the cotyledon was suppressed greatly if an excised

plumule or radicle of a mature zygotic embryo was placed in contact with the cotyledon.

indicating that embryogenesis was suppressed by endogenous inhibitors present in the axis

tips of the mature zygotic embryo. Ham and Lee (1974) reported that callus and organized

structures formed from ginseng cotyledon explants cultured on medium without plant growth

regulators, and subsequently grew into plantlets. Somatic embryos were produced on the

cotyledons of 30% zygotic embryos on medium without plant growth regulators (Lee et al.,

1990). In order to obtain high frequency of SE production in a short t h e , 2,4-D (5 PM)

combined with BA (0.1 PM) and zeatin (0.1 pM) or, 3,4-D (5 PM) combined with TDZ

(0.01 pM) were used for both seedling pretreatment and tTCLs culture (Ah et al., 1996).

The fiequency of embryogenesis decreased as the concentration(s) of cytokinin(s) increased.

On the other hand, globular- and heart-stage SES developed into cotyledonary-stage embryos

on MS medium containing NAA (0.3 PM) / BA and zeatin ( 1 pM) in 4 weeks but failed to

grow M e r ( A h et al.. 1996).

MS medium enriched with auxins (IAA; indole-3-butyric acid, IBA; NAA or 2,4-D).

BA and gibbereliic acid (GA,) were used for callus culture of young flower buds of ginseng

(Shoyarna et ai.. 1988). High-yield (75%) embryogenesis via callus was induced on 4.5 pM

2.4-D supplemented medium in the dark within 3 months. Kishira et al. (1 992) induced SES

fiom young flower buds of both in vitro culture and 5-year-old field grown plants. With in

vitro matenals, SE fomed at a frequency of 80% afier 3 months of culture on MS plus 3.4-D

(4.5 PM). Young flower buds from field grown plants, gave higher fiequency (90%) of

embryogenesis on 22.5 FM 2,443 than 4.5 pM (55%) or 45 pM (75%) 2,4-D after 178 day

of incubation.

Somatic embryogenesis at a frequency of 30% has been obtained in adventitious

shoot culture on MS medium containing 4.5 pM 2,4-D (Kishira et al., 1992). On the other

hand, embryo-like structures in various stages of development appeared on leaf-derived

callus after 6 months of culture on NAA (10.7 FM) and kinetin (2.3 PM) enriched LS

medium (Linsmaier and Skoog, 1965) under 16 h-photoperiod (Celliirova et al., 1992). Many

anomaious structures were observed, such as two SES of different stages of development had

same ongin, or SES had asymrnetncal cotyledom. If embryogenic callus were maintained on

the induction medium, leaf-like structures and rhîzoids formed. When these calli were

transferred ont0 basai medium. secondary SES developed, especidly on the surface of

cotyledons and leaf-like structures.

Chang and Hsing (1980) induced ginseng callus by culturing mature roots on MS

medium emiched with lg / L casein hydrolysate and 4.5 pM 2.4-D in the dark at 26°C.

Callus formed after 3 weeks of incubation, and globular and heart-shaped SES were observed

on 8-months-old callus. These SES were white in colour, bipolar, and loosely attached to

the callus. Abnormal cotyledonary SES (e-g. poly-cotyledonary SE). were cornmonly

observed. In addition, some of the SES did not go through the normal developmentd stages,

but instead, they swelled, callused and gave rise to secondary SES.

Choi et al., (1982) also induced SE from root callus using a two-step procedure.

Roots of 6-year-old plants were incubated on modified MS medium containhg with 0.45-36

pM 2,4-D for callus induction, and then root calli produced both SES and shoots in 112 MS

medium supplemented with 10.8 pM NAA and 9.9 FM N~ (&isopentenyl)-adenine (2iP)

after 5 months of culture. Various malformed SES were observed.

Furuya et al., (1 986) reported adventitious shoot or root formation fiom callus culture

of 5-year-old root explants. The callus was induced on MS medium contain 4.5 FM 2,4-D

and 0.46 pM kinetin, and maintained on the same medium at 20 O C in the dark for 5 years.

A t-igid and compact callus appeared when the original callus was transferred to MS basal

medium. while supplementing the medium with 4.6 p M kinetin stirnulated the development

of multiple green shoots, and roots were induced when the medium was supplemented with

4.9 pM IBA in the dark. In addition. SES were induced from those shoots with the treatment

of high temperatures and hi& sugar concentrations (Asaka et al., 1993; Asaka et al., 1994a).

Somatic embryos have dso been produced in protoplasts cultures isolated From

pnmary SES derived fiom zygotic embryos (Arya et al., 1 99 1 ). MS medium supplemented

with 4.5 pM 2,4-D combined with 0.05 p M kinetin was used for SE formation.

1.4.1.2 Somatic embryo development and plant regeneration (Table 1.4)

Chang and Hsing (1980) showed that when SES derived nom mature roots were transferred

ont0 either 54 MS or BI medium (Gamborg et al., 1968) containing 4.4 pM BA and 2.9 p M

GA,, they developed etiolated shoots in the dark and normal green shoots in the light. The

same combination of GA, and BA has d s o been used for plantlet formation from SES

denved fiom zygotic embryos (Lee et al., 1990), Agrobucterium transforrned cotyledons (Lee

et al., 1995), and protoplasts derived SES (Arya et al., 199 1). Somatic embryos with multi-

cotyledons and plantlets with multiple shoots have ofien been observed (Arya et al.. 199 1).

Intact plantlets developed from flower bud derived SES on MS-vermiculite medium

containing 1.45 pM GA, and 2.2 pM BA under 16h-photopenod at 16 O C (Kishira et al.,

1992). The average length of plantlets was 8 mm after 4 weeks of culture. In addition. the

shoot growth was inhibited when SES were cultured in 1/2 MS liquid medium containing

1.45 pM GA,. -1

In order to get shoot formation from SES obtained fiom young flower bud-derived

Shoyama et al., (1 988) used 1.5 or 3 % sucrose in the % MS medium containing 1 -4

p M GA, and 2.2 pM BA. The rates of normal shoot regeneration were 7 1 % at 1.5% sucrose

medium compared to 45% at 3% sucrose level. On the other hana root induction was tested

in MS medium supplemented with various auxins including NAA, [BA, or IAA, and 75%

of shoots treated with NAA (5.4 pM) developed into rooted plantlets. Plantlets were

subsequently transplanted into vermiculite at 20 O C and cultured under controlled humidity

for one rnonth. The transplantation rate was over 70%. However, ~e l l i rova et al. ( 1992) were

not able to induce roots utilizing the same protocol, but they obtained shoots with SES from

leaf-derived callus.

On MS medium supplemented with 4.6 FM kinetin, cotyledonary stage secondary

SES formed shoots under 16 h-photoperiod at 20 OC in 5 weeks (Arya et al., 1993), while

roots formed on MS medium containing 4.6 p M kinetin and 2.9 p M GA,. Secondary SES

ais0 developed into plantlets when cultured on % MS medium with 4.4 FM BA (or 4.6 pM

kinetin) combined with 2.9 FM GA,, although the fiequency of plantlet formation was lower

(60 %) compared with that (80%) using the two step procedures.

1.4.1.3 In vitro flowering (Table 1.4)

Chang and Hsing (1980) induced well-stmctured flowers in vitro fiom root-derived SES

without establishing normal seedlings. The cultures were incubated on B, medium containing

4.4 pM BA and 2.9 FM GA, at 26 OC under 16 h-photopenod. The authors found that 90%

of the pollen grains were fertile, and suggested that the SE system could provide materials

for studies of flowering without the need for the normal juvenile phase of about 3 years.

Shoyama et al.. (1988) showed flower formation from the SES derived from young

flower buds cultured on 4.4 p M BA and 1.4 pM GA, enriched 1/2 MS medium. Lee et al.,

(1995) reported that more han 50% of the plantlets derived fiom cotyledon-induced SES

flowered in viiro after 10 weeks of culture on 112 MS medium supplemented with GA, (2.9

PM) and BA (4.4 FM). Wiîh same medium and growth regdaton, Zygotic embryo-denved

SES flowered under both dark and 16 h-photopenod (Lee et al., 1990). Three to 15 flower

buds developed on an umbel, although flowers were much smaller than those on the field-

grown plants. A few immature fniits were also observed in vitro.

In vitro flowenng was also induced fiom zygotic embryos (Lee et al., 1991).

Although flowers obtained in viiro were several-fold smaller than those in situ, some of

them bore immature fniits which were about 5 mm long. Reducing the strength of MS salts

to half enhanced the fiequency of flowering. Furthemore, BA was necessary for in vitro

flowering, with 5 FM as the optimal concentration which induced a flowenng frequency of

80%. In addition, abscisic acid (ABA) could block the BA-induced flowering process, and

GA, was essential for reversal of the inhibitory effect of ABA. GA, alone was found to be

not sufficient to induce in vitro flowering.

1.4.2 American ginseng

1.4.2.1 Callus induction and somatic embryogenesis (Table 1.5)

Callus and somatic embryos of American ginseng have been induced From zygotic embryos

(Gui et al., 1987; Li and Guo, 1990), epicotyls and leaves (Tirajoh and Punja, 1995), and

roots (Wang, 1990; Tirajoh and Punja, 1995).

Gui et al., (1987) showed that zygotic embryos £iom stratified seeds gave rise to

callus and SES in MS medium containing 2.3 PM 2,4-D. SES at different stages were often

a e

found on the same explant. In addition. many abnormal SES were observed. although they

were bipolar structures, and easily isolated fiom the callus tissue.

Li and Guo (1990) used various types of materials for sornatic embryogenesis

including zygotic embryos (from der-ripened seeds) and etiolated seedlings (from after

npened seeds which germinated in a refngerator to 5 cm in height), and main roots and

petioles (both fiom 4-year-old plants). Callus was induced fiom al1 type of explants, but

SES were only observed on embryo-derived callus. and occasionally on etiolated seedling-

derived callus. The medium used for zygotic embryo explants were MS or B, medium with

2 , 4 D (4.5 or 9 pM). A nurnber of embryos at various stages emerged on the surface of the

soft calii derived fiom swollen cotyledons and hypocotyls after 6 weeks of incubation. Both

B, and MS medium were effective for inducing callus, while B, medium alone stimulated

the formation of adventitious roots.

Root explants fiom 4-year-old field-grown plants, formed compact callus on MS

medium containing 9 p M 2.4-D and 4.6 pM kinetin, and became friable when the kinetin

was removed from the medium (Wang, 1990). Embryogenic callus developed when 9 pM

3,6-dichloro-O-anisic acid (dicarnba) was used instead of 2,4-D. The author indicated that

Amencan ginseng develops more rapidly on a medium with dicamba, in contrast to Oriental

ginseng, which grows better on a medium containing 2,4-D. Similady, 4-5-year-old field

grown roots grew better on MS medium containing dicamba and kinetin than on 2,4-D and

kinetin containing MS medium (Tirajoh and Punja, 1995), and subsequently formed SES

d e r 6 months of culture in the dark. The most effective combination for optimal growth of

callus in the first 3 months was 9 pM dicamba and 5 p M kinetin.

Epicotyls (fiom in vitro grown seedling) and leaves (both fiom 4-5 year old field

grown plants and in vitro grown seedlings) have been used for SE induction (Tirajoh and

Punja, 1995). In vitro grown seedlings were obtained by growing seeds (stratified in cool-

warm-cool temperature for 9 to 12 months) on MS medium containing BA (4.4 PM) and GA,

(2.9 PM), and subculniring at monthiy intervals until they germinated in 4 to 6 months.

Epicotyl explants were cultured on MS medium containing dicamba and kinetin, and

somatic embryos formed in 9 months of incubation at 2426°C in the dark (Tirajoh and

Punja, 1995). Leaf explants were cultured on MS medium supplernented either NAA (5. 10'

15 PM) with 2,4-D (9 FM) or dicarnba (4.5,9.0, 13.5 pM) with kinetin (5 PM). SES formed

on NAA and 2,4-D enriched medium from seedling-denved callus in 3 months, and from

field-grown plants in 6 months. The tirne required for SE production and the nurnber of SES

formed was negatively influenced by leaf age. In addition, no SES formed on dicamba and

kinetin supplemented medium until the cailus was msferred to the medium with NAA and

2.4-D. Also, callus maintained under light (16 h- photoperiod) turned greenish-purple in

colour. and no embryogenic callus was observed even when subsequently placed in the dark.

They suggested that dark initiation was required for SE formation (Tirajoh and Punja, 1995).

1.4.2.2 Somatic embryo development and plant regeneratioo (Table 1.6)

The processes o f plantlet conversion have been studied by several researchers, and shoots

(Tirajoh and Punja. 1995) and plantlets (Gui et al., 1987; Li and Guo. 1990; Wang, 1990)

have been obtained.

MS medium containing 4.4 p M BAP and 2.9 FM GA, were used for germination of

SES obtained fiom root, leaf and epicotyl-derived callus (Tirajoh and Punja, 1995). shoots

were fomed in one and half months of incubation under 1 oh-photoperiod. but root formation

has absent.

Cotyledonary stage SES derived from zygotic embryos developed into plantlets on

MS medium e ~ c h e d with BA, NAA and other plant growth regulators (Gui et al., 1987).

Li and Guo (1990) showed that plantlets can be induced fiom zygotic embryo explants

derived SES on B, medium supplemented with 0.9 pM 2,443.2.9 pM GA, and 1% sucrose.

Wang ( 1990) developed the procedure to obtain plantlets following SE development

and maturation. The embryogenic calli obtained from roots were transferred ont0 an embryo-

maturation medium that was composed of MS salts. B, vitamins, NAA (2.2 pM). 2,4-D (4.5

PM), sucrose (30 mg/ L) and agar (7 g/ litre) and maintained at 27 O C with 1 6 h-photoperiod.

The matured SES (in about 3 weeks) were transferred to MS medium containing various

plant growth regulators including BA, GA. IBA. kinetin and NAA . and incubated at 27 OC

and 16 h-photoperiod. Normal plantlets regenerated on medium supplemented with 2.5 pM

IBA and 0.54 pM NAA. The frequency of regeneration was 30%.

In surnrnary, induction of somatic embryos and plant regeneration from somatic

embryos in Oriental ginseng have been reported by many researchers. However, in

American ginseng, only a few published information are available to date. Furthemore,

the thne p e n d required for somatic embryogenesis was long (Tirajoh and Punja, 1995),

and the embryogenic frequency is still unknown (Gui et al., 1987; Li and Guo, 1990;

Wang 1990). In addition, the ginsenoside content of regenerated plantlets has not been

docurnented and no plantlets derived from SES have k e n transplanted into soi1 or soilless

mixture.

The objectives of this study were:

1) to induce somatic embryos of Arnerican ginseng at hi& frequency, and study the effect

of plant growth regulators. and type of explant on embryogenesis;

2) to produce plantlets fiom somatic embryos at high conversion rate;

3) to analyse ginsenoside level in regenerated plantlets.

Table 1.1 Ginsenoside content of callus and regenerated plants from somatic embryos of

Oriental ginseng ( P u n a ginseng C. A. Meyer) (Asaka et al., 1994)

Materiais Total content per dry mas# Ratio of Rb to Rg (wt %)

Callus (3% sucrose! 0.15 0.83

Callus (8% sucrose) 0.09 0.77

Somatic embryo" 0.72 0.65

Shoot (plantlet?

Shoot (seedling )

Root (plantlet) 0.30 0.79

Root (seedling) 0.23 0.72

ITotal content was calculated from the quantity of detected ginsenosides by KPLC,

including Rb and Rg groups.

'MS medium containing 3% sucrose.

"Somatic embryos were obtained fiom multiple shoots treated with high temperature and

high concentration of sucrose.

"Shoots of plantlets regenerated from somatic embryos.

Table 1.2 Ginsenoside content of original plants and tissue culture matenals of Oriental

ginseng (Panax ginseng C. .4. Meyer) (Furuya et al., 1 986)

Materials Total content per dry m a s z Ratio of Rb to Rg (wt %)

Multiple shoots"

Adventitious roots" (callus culture)

Adventitious rootsv (suspension culture)

Shoots (original plants)

Roots (original plants)

'Total content was calculated fiom the quantity of detected ginsenoside by TLC method.

including Rb and Rg group.

Tallus was induced on MS medium containing 2.4-D (1 FM) and kinetin (O. 1 PM) fiom

5-year-old root.

'Adventitious shoots were induced on kinetin ( 1 PM) containing medium.

"Adventitious roots were induced on IBA (1 FM) containing medium.

'Adventitious roots were induced on IBA (2 PM) and kinetin (0.1 PM).

Table 1.3 Somatic embryogenesis in Orientai ginseng (Punar ginseng C . A. Meyer) - - - --

Rrsponse Basal Media 'PGRs (PM) Explant Reference

Somatic embryo (SE)

Ahn et al., 1996

Callus and SE

Callus and SE Z~gotic embryo Aqa et al.. 1993

SE

Cal lus and SE

Adventitious

Leaf

Asaka et al.. I 994

Cellirova et id.. 1992

Chang and Hsing. 1980

Choi et ai.. 1982

Choi et ai.. 1982

Cailus and SE Root

Callus'

SE"

Roo t

Callus

Cotyledon

Callus

Choi et al.. 1984

Choi et al.. 1982

Zygotic embryo. cotyledon

Choi et al.. 1996

Furuya et al.. 1986 Root

Root. stem. and leaf Jhang et al,. 1974

Callus and SE Adventitious shoot Young flower bud

Kishira et al.. 1992

Callus and SE Zygotic embqo Lee et al.. 1990

Lee et al.. 1995 Callus and SE (0.46) (transformed)

'Plant growth reguiator(s).

ytransverse Thin Cell Layers.

"Two step process has been used. callus induction on 2.4-D (22.6 PM) medium. and sornatic embryo formation on

NAA ( 10.7 PM) and 2iP (9.8 PM) medium.

"A two step process.

Table 1.4 Plant regeneration from somatic embryos in Oriental ginseng (Panax ginseng)

Response Basal Media TGRs (PM) Reference

Plantlet

Shoot

Root

P lantlet

PIantlet

Shoot

Shoot. in vitro flowering

Plantlet, in vitro flowenng

Plantlet, in vitro flowering

Plantlet (limited shoot growth)

Shoot

Root

In vitro flower

MS

MS

MS

112 MS

MS

1/2 MS

1/2 MS or B5

112 MS

112 MS

MS- venniculi te

1/3 MS (1 iquid)

1 2 MS

Y

112 MS

GA, (2.9). BA (4.4)

Kn (4.6)

GA, (2.9), Kn (4.6)

BAKn (4.4/4.6), GA, (2.9)

-

GA, (1.4), BA (2.2)

GA, (2.9), BA (4.4)

GA, (2.9), BA (4.4)

GA3 (2.9), BA (4.4)

GA, (1.45). BA (2.2)

GA, ( 1.45)

GA, (1 A), BA (2.2)

NAA (5.4)

GA, (1.4). BA (1 1.1)

Arya et al., 199 1

Arya et al., 1993

Arya et al., 1993

Arya et al., 1993

Asaka et al., 1994

Celliirova et al., 1992

Chang and Hsing, 1980

Lee et al., 1990

Lee et al., 1995

Kishira et al., 1993

Kishira et al., 1992

Shoyarna et al.. 1988

Shoyama et al., 1988

Shoyama et al., 1988

'Plant growth regulator(s).

'net indicated.

Table 1.5 Somatic embryogenesis in Amencan Ginseng (Panax quinquefolizrm L.)

Response Basal TGR(s) (PM) Explant Reference

Zygotic embryo

Leaf, petiole, stem, root

Gui et al,, 1987

Jhang et al., 1 974

Callus and SE

Callus

Callus and SE

CalIus and SE

Callus and SE

Callus

Callus

Callus

Callus

Callus

CaIIus

Zygotic embryo

Zygotic embryo

Li and Guo, 1990

Li and Guo, 1990

Li and Guo, 1990

Li and Guo, 1990

Li and Guo. 1990

Li and Guo, 1990

Li and Guo, 1990

Li and Guo. 1990

Li and Guo. 1990

Zygotic embryo

Hypocotyl

Hypocotyl

Hypocoty 1

Hypocoty 1

NAA (54) Hypocoty 1

Petiole 2.4-D (4.5), NAA (5.4)? Kn (2.3)

Callus and SE dicarnba (9). Kn ( 5 )

Epicotyl. root Tirajoh and Punja 1995

Callus and SE NAA (5.10, 15), 2.4-D (9),

Leaf Tirajoh and Punja 1995

Root Wang, 1990

SE" . Wang, 1990

'Plant growth regulator(s).

Y"A two step process has been used, callus induction occurred on 2,4-D (9 FM) plus

kinetin (4.6 FM) medium, and SE induction occurred on dicarnba (9 FM) containing

medium.

dicarnba (9) root-callus

Table 1.6 Plant regeneration fiom somatic embryos in Amencan Ginseng

- -- -- - - - - - -- - - - -

Response Basal Media TGR(s) (PM) Reference

PIantlet MS BA, NAA and Gui et al., 1987 other PGRs

Plantlet B5 2,4-D (0.9), GA, (2.9) Li and Guo, 1990

Shoot MS GA, (2.9), BA (4.4) Tiraj oh and Punj a, 1995

Plantlet MS NAA (0.54), Wang, 1990

'Plant growth regulator(s).

Chapter 2. Induction of somatic embryos - Effects of pretreatment,

sucrose concentrations, auxin type and concentration on different

types of explants of Arnerican ginseng

2.1 Introduction

Auxins are required for production of embryogenic callw in many plant species (Amirrato,

1983; Kysely and Jacobsen, 1990; Lazzeri et al., 1987a b). The study of somatic

embryogenesis in carrot (Schiavone and Cooke, 1 987; Michalczuk et al.. 1 992) revealed that

auxins play important roles both in the initiation and development of somatic embryos (SES).

In comparative studies of different auxins, 2,4-D was very effective, being used widely in

both angiosperms and gyrnnosperms for induction of callus and SES (Brown et al.. 1995;

Dunstan et al.. 1995: KrishnaRaj et al., 1995). NAA and dicamba are two other auxins which

have also been used for callus and somatic embryo (SE) induction. Different auxins are not

equal quantitatively or qualitatively in their actions (Baker and Wetzstein. 1994; Banvale et

al.. 1986; Tabei et al., 1990). In addition. concentration of auxin was a critical factor

controlling somatic embryogenesis (Choi et al., 1996; Grifin and Dibble, 1995; Jia and

Chua, 1992). Furthemore, dif3erent responses of various explant types for auxin have been

reported in Allium (Havel and Novak, l988), Chinese cabbage (Choi et al., 1 996). Cyclamen

(Takamura et al., 1995), and sugarbeet (Tetu et ai., 1987).

Although the information on the effect of light and GA, as pretreatments for seedling

growth on subsequent somatic embryogenesis is lacking, light as an environmental factor

moderates the embryogenic process (Gray et al., 1993; Lu et al., 1984; Reynolds and

Crawford, 1997). The effect of GA, on embryogenesis have also been shown (Chen et al.,

1987; Hunault and Mautar, 1995; Kao et al., 198 1 ; Hutchinson et al., 1997). Concentration

of sucrose is another key factor influencing the process of embryogenesis (Binzel et al.,

1996; Laneri et al., l987b; May and Tngiano, 199 1 ; Michler and Bauer , 199 1).

In ginseng, 2.4-D has been used in both Oriental (Chang and Hsing, 1 980: Jhang et

al., 1974; Kishira et al., 1992) and Amencan ginseng (Gui et al., 1987; Li and Guo, 1990;

Jhang et al., 1 974). as well as in Panm notoginseng (Shoyama et al., 1997) and Panax

japonicus (Shoyama et al., 1995) for production of cailus and somatic embryogenesis. NAA

has been used effectively for induction of callus from hypocotyl explants of American

ginseng (Li and Guo, 1990), even though no SES were formed, whereas SES were induced

on leafexplants obtained from in vitro grown seedling after 3 months of culture on 2.4-D and

NAA supplemented medium (Tirajoh and Punja, 1995). In addition. SES have been

produced on dicamba containing medium from root derived callus (Wang, 1990). However.

no comparative study of the effect of different types of auxin and their concentration on

somatic embryogenesis in American ginseng has been reported, and the effect of

pretreatment on SE formation is unknown.

The objectives of this work were to study:

1) the effect of auxin(s) on somatic embryogenesis in various types of explants from

stratified seed;

27

2 ) the effect of pretreatrnent for seedling growth of both statified and green seeds on

subsequent embryogenesis;

3) the influence of sucrose concentration on somatic embryo induction.

2.2 Materials and rnethods

Seed source

Stratified seeds, which were used in al1 experiments in this chapter, had the traditional

stratification process (mixed with sand and stored in sandbox outdoor for one year, Proctor

and Louttit, 1995) were purchased fiom commercial growers in Waterford, Ontario, in the

Fall. M e r receipt, seeds were size graded, and extra large ( > 5.95 mm diameter) and extra

small (< 5.16 mm diameter) seeds were removed. n i e rest of the seeds were mixed with

moist sand. and stored in a cooler (3 * 0.2 OC) until used. "Green" seeds , which were used

in the "pre-treatment" expenment did not have the traditional stratification process, were

purchased in the Fall, graded and mixed with rnoist sand as described above, and then stored

at 15 0.2"C for 3 months before tramferring to 3 O C until used (Hovius, 1996).

For experirnents. seeds with cracked seed coat (Fig. 2.1A) were taken out of the

cooler. the sand washed away, and the seed surface sterilized by immersion in 40%

commercial bleach solution (2.2% sodium hypochlorite) with Tween 20 (two drops per 100

ml solution) for 10 min with continuous stimng. The seeds were then rinsed for 3-5 times

with sterile distilled water. and the seed coat was removed.

M e r removing the seed coat, seeds were nnsed with distilled water then immersed

in 70% ethanol for 2 min, and re-sterilized with 2.2% sodium hypochiorite for 20 min while

stimng, followed by at least 5 washes with sterile distilled water.

fiplant iype

Three types of explants were tested:

1) Zygotic embryos (ZEs) - intact mature embryos (4-5 mm long) excised £iom stratified

seed. Mature and immature ZEs are shown in Figure 2.1 B and D,

2) Cotyledons - isolated cotyledons of in vitro grown seedling (Fig. 2.1 C, 2.2 and w t i c

embryo germination in this chapter). Al1 segments of seedlings, Le., apex and stem,

cotyledons. and root, were used in some of experiments as well.

3) Shoots - shoots obtained from somatic embryos. The procedure is described in Chapter

4 (see Fig. 4.1 ).

Zygotic embryo germination

The endosperm of the seed was cut open aseptically under a dissecting microscope, and the

zygotic embryo (ZE) was excised and cultured in a Petri dish. The germination medium for

ZE culture was 1 p M gibberellic acid (GA,) contained basal medium (BM) which consisted

of MS (Murashige and Skoog. 1962) salts, B, vitamins (Gamborg et al., 1968). and 30 %

(w/v) sucrose. Gelnte (0.25 %) (Scott Laboratories, Carson. USA) was used as the gelling

agent. The pH of the medium was adjusted to 5.6 using O. 1 N and 1 N of each of NaOH and

HCI. The media were autoclaved at 1.19 kg cm-' for 20 min, cooled down, then poured into

pre-stenlized Petri dishes (1 00 x 25 mm; Fisher Scientific Co.' Unionville, Ontario). Cultures

were maintained in a growth room at 24 OC under 16h light period (60 pmol m-' s-'; cool

white fluorescent larnps; Philips, Scarborough, Ontario) for one week.

Callus and somatic embryo induction

The induction medium consisted of BM, or BM supplemented with various types and

concentrations of auxin(s). The pH of the media was adjusted to 5.6 before autoclaving. Al1

the cultures were incubated in a growth room in the dark at 24 OC. The following

experiments and pre-treatment schedules were developed fiom suggestions by Gui et al..

(1987) and Li and Guo (1990) and were used to identifi optimal conditions for callus and

SE induction,

1. Effect of auxin(s)

a) To compare three types of auxins, BM with or without 2,4-D (1.5. 10, 15 FM), NAA (5,

10, 15 FM) or dicamba (5, 10. 15 PM) was used for culturing ZE and cotyledonary explants.

The sarne auxin(s) were used for shoot explants except that the concentrations of 2,4-D used

were 1,2, 5. and 8 FM.

b) To test the effect of culture duration on 2,4-D containing medium. 5 pM 2,4-D was used

in BM for 0, 2. 4. 6. or 8 weeks before being removed. Three types of explants: ZEs.

cotyledons. and shoots. were tested.

C) To test the effect of combination of 2,4-D and NAA, nine combinations of 2.443 (5, 10.

and 15 FM) and NAA (5,10, and 15 pM) were used with ZEs, cotyledon, seedling segments,

and shoot explants.

2. Effect of pretreatment

To test the effect of pretreatrnent on subsequent somatic embryogenesis, various

3 1

concentrations of GA, (O, 1, 5, 10 FM) were added to medium for ZE germination. the

cultures were kept either in the dark or 16h light for both stratified and green seeds. The

cotyledons of one-week-old seedlings were then used as explants which were culnired on

BM containing 5 PM 2.4-D and 15 FM NAA for SE induction (Fig. 2.2).

3. Effect of sucrose

Sucrose at concentrations of 1.5,3.0,6.0, and 9.0 % were used in BM for shoot explants, and

the cultures was kept in light or in darkness. Explants were either kept in various

concentrations of sucrose for 8 weeks or transferred to 3.0 % sucrose after 2 weeks of

cul tue.

Sucrose at concentrations of 0, 1.5, 3, 6, 9. and 12 % in BM was used for ZE

explants. Explants were either kept in various concentrations of sucrose for 8 weeks or

transferred to 3.0 % sucrose after 2 weeks of culture. A11 the cultures were incubated in

darkness.

Data colleet ion and analysis

In most experiments, four explants were placed in one Petri dish with ten dishes (replicates)

for each treatrnent. and each experiment was repeated at l e s t twice.

Cultures were exarnined under a dissecting microscope weekly. The time required

for SE induction represeiits the period preceding the emergence of SES. Embryogenic

fiequency (percentage of the explants inducing SES) and the number of SES produced in each

dish were recorded after 8 weeks of induction, and presented as mean *standard error. A chi-

square test was used to do the trend anaiysis for the embryogenic frequencies produced with

32

different levels of growth regulators. Log transformation was carried out for the data of

number of SES, and then the analysis of variance was calculated using the General Linear

Mode1 (GLM) procedure of the Statistical Anaiysis System (SAS Institute, 1985). Back

transfo rmed data are presented. The occurrence of adventitious roo ts and bud-like structures

was also recorded.

Histo/ogy

Calli with SES were harvested and cut into pieces of about 0.4 cm' and fixed in FAA (50%

ethanol: 10% formalin : glacial acetic acid; 18: 1: 1) for 24 h at room temperature. The

explants were then dehydrated in a graded senes of ethanol(50, 70, 80, 95, and 1 00%), and

embedded in paraffin using a protocoi similar to that descnbed by O'Brien and McCully

(1 98 1).

The parfiln blocks were trimmed and mounted on to a wooden base. The mounted

blocks were held in the cold (5 + 0S0C) for one day to harden, then trimmed into a trapezoid

shape with even, parallel faces. Ten FM thick sections were obtained by sectioning with a

microtome (Spencer 820, Arnencan Optical Co., New York). The sections were mounted

on glass slides with Haupt's adhesive. The slides were piaced on a warm tray (27-30°C) for

1-2 d to allow the sections to expand.

The process for deparaffinking was similar to that outlined by Johanson (1940).

Sections which went through 100 % Hemo-De, 50: 50 100 % Hemo-De: 100 % ethanol and

a graded series of ethanol from 100,95, 70, to 50 %. The specimens were then stained with

Alcian Green / Safranin Alcian for half hour, Mised with water, then put through a series of

33

ethano1 from 50,70,90,95. to 100 %, and eventually to 100 % Hemo-De. Permanent slides

were made with Eukitt. Representative sections were photographed with a Zeiss Universal

photomicroscope (Car1 Zeiss. Oberkochen, Germany).

Scanning elecfron microscopy (SEM

The procedure of sarnple preparation was similar to that descnbed by Massicote et al.,

(1 987). Somatic embryos at various developmental stages were isolated from callus tissue,

and fixed with 4% glutaraldehyde in 0.07M phosphate buffer at pH 6.8 for 3-24 h at room

temperature with occasional shaking (Massicote et al., 1987). The waste glutaraldehyde was

then removed with a Pasteur pipette, and the SES were washed twice in phosphate buffer. 5

min each. Fixed SES were then dehydrated in a graded series of ethanol, starting with 30%

for one and half hour, then 50,70,90,95, and 100 % each for a half hou. The 100 % ethanol

was changed twice, one hour for each time, and then the samples were stored in 100 %

ethanol at room temperature until used. Dehydrated samples were criticai- point dried,

rnounted on stubs with two-sided tape, then sputter coated to 30 nm thickness with gold /

palladium (Anatech. Hummer VIII, Alexandna, VA). and examined under a JEOL JSM-3%

or Hitachi S-570 (Tokyo) scanning electron microscope.

2.3 Results

1. Effect of auxin(s)

a) Cornparison of 3 types of auxin

Co Nedonary explants

Over 80 % of the explants on BM tumed brown, aithough some callus but no SES were

observed in a few explants. SES at different stages of development were observed with

explants incubated on auxin(s) supplemented medium. and SES with multi cotyledons were

also found.

The highest frequency (43 %) of embryogenesis on 2,4-D was observed at 5 p M

(Table 2.1). Ten and 15 pM 2,4-D gave dark yeilow callus and a few small SES in the

globular stage. Both fiequency and number increased linearly with the 3 concentrations of

NAA tested with the frequency and number of SE per dish at 47 % and 8. respectively. at 15

FM NAA. Also. 1-1 0 roots per explant were counted with medium containing NAA (Fig.

2.3A). and in general. more roots were found with higher concentrations. Dicarnba at 5 pM

gave the highest fiequency (25%) of embryogenesis among the concentrations tested, and the

fiequency was reduced dramaticaily as the concentration increased (Fig. 2.3B).

Zygotic cmbryo explant

Most zygotic embryos germinated on BM (Fig. 2.4A), but no SES were found. Some callus

formed on cotyledons and on the base of the embryos with 2,4-D containing medium (Fig.

2.4C). Five FM 2.4-0 gave the highest fiequency (12 %) of SE induction among the

concentrations tested (Table 2.1). Some callus, adventitious roots and a few SES were found

in embryos cultured on NAA containing medium (Fig. 2.4B), and the highest frequency was

22 % at 15 pM. Dicamba e ~ c h e d medium gave only O - 3 % fiequency of SE formation.

and elongated and curled shoots were often observed (Fig. 2.4D).

Shoot esplants

Most explants on BM were dark brown in colour (Fig. MA) , and only a few explants had

some callus but no SES. At 1 and 2 pM 2,PD a similar fiequency of 52 -54 % was obtained

and some high quality SES which were in the cotyledonary stage and separate from each

other (Fig. 2.5B). Higher concentrations of 2,4-D fomed callus which had a watery-

appearance, with lower frequency and small SES (Table 2.2, Fig. 2.5C). Adventitious roots

were observed with explants on NAA containing medium, and similar fiequencies (50-58

%) of SE induction for ail the concentrations tested (Fig. 2.5D). Good quality SES were

observed on the explants cultured on 5 pM dicamba which gave the highest fiequency (45%)

and nurnber arnong the concentrations tested.

In general, the response to 2,4-D and dicamba exhibited a similar quadratic trend: 5

pM gave a better response than lower or higher concentrations, except in the case of shoot

explants. in which 1 or 2 pM gave a better response than 5 or 8 FM. In NAA containing

medium, however, the frequency and number increased linearly as the concentration

increased fiom 5 to 15 FM.

Among the 3 types of explants, shoots derived from SES showed the best response

and ZE the leut. Also, shoot explants required a lower concentration of 2,4-D to give high

fiequency and number of SES induced than cotyledonary or ZE explants.

b) Effect of 2'4-D and NAA

Concentrations of 2,4-D (5, 10, 15 PM) significantly influenced ernbryogenesis in al1 the

explants tested, and the effect of 2.4-D was independent of the concentrations of NAA (Le..

no interaction between 2,4-D and NAA). Therefore, the main effects of 2,4-D and NAA are

presented.

Coryledonary explants

About 58 % of the explants cultured on 5 pM 2,4-D supplemented medium gave rise to SES

compared to 38 % and 27 % observed on 10 and 15 FM 2,4-D, respectively (Table 2.3. Fig.

2.6A, B). The three concentrations of NAA used did not affect the frequency of

embryogenesis and the number of SES produced.

Seedling segments

The optimal concentration of 2.4-D was 5 PM. giving a frequency of 27 % and 6 SES per

dish (Table 2.3). The fiequency reduced from 23 to 14 % as the NAA levels increased from

5 to 1 5 FM. In addition, 98.5 % of the total number of SES induced in seedling segments

were formed on cotyledonary segments whereas shoot and root segments only formed 0.6

% and 0.9 % of the total SES, respectively (Table 2.4).

Zygotic emhryo explants

The frequency of SE induction and SE number per dish were affected significantly by 2.4-D

but not by NAA. The optimal treatment was 5 FM 2,4-D which induced SES fiom 1 1 % of

the explants (Table 2.3). The number of SES per dish was low in al1 treatments. and some

SES with multiple cotyledons were found (Fig. 2.6C).

Shoot explants

The highest fiequency (62 %) was recorded on explants cultured on 2 FM 2,4-D

supplemented medium whereas explants on 5 and 8 pM 2,4-D containing medium gave a

fiequency of 36 % and 24 %, respectively (Table 2.3. Fig. 2.7). The three concentrations of

NAA used did not affect the response significantly.

c) Effect of exposure time to 5 FM 2,4-D supplemented medium

Cotyledonary explants

Over 70 % of the explants which had no exposure to 2 ,4D were brown in colour (Fig.

U A ) . no SES but some callus fonned in two explants. From 34 - 47 % of the explants

which had exposure to 2,4-D gave rise to SE (Table 2.5). Exposure time of 4 or 6 weeks

gave a higher frequency than 2 or 8 weeks . Adventitious roots were observed in 50 % of the

explants which had 2 or 4 weeks exposure to 2,4-D, and the SES which formed in explants

which had 2 weeks exposure to 2,4-D were often fused together and to the callus (Fig. 2.8B).

On the explants which were maintain on 2,4-D containing medium for the whole 8 weeks

penod of induction, SES at various stages of development were observed (Fig. 2.8C).

Zygoric ernbryo explants

About 90 % of the explants which had no exposure to 2.4-D germinated with the seedling

length up to 50 mm, but no callus or SES were found. Oniy 2.6 % of the explants gave rise

to SES after 2 weeks exposure. which was significantly lower than the fiequency (1 0 - 15 %)

observed with other treatments (Table 2.5). The number of SES per dish was low in ail

treatments. About 20 % of the explants formed adventitious roots.

Shoor explants

Although most explants which had no exposure to 2.4-D were brown in colour, 4.2 % of the

explants gave rise to SES with two SES per dish (Fig. 2.9A). while other treatments exhibited

a fiequency of 52 - 68 %, and up to 42 SES per dish were recorded (Table 2.5. Fig. 2.9 B. C).

Exposure time of 2-6 weeks gave a higher fiequency than 8 weeks. SES formed on explants

which had 2 weeks of exposure were of lower quality (fused embryos) than those formed on

explants which had longer exposure time to 2.4-D. in addition, 39 - 56 % of the explants

which had 2 - 6 weeks of exposure to 2.1-D formed adventitious roots.

Comparing the 3 types of explants in the experiment, high frequencies of SE

induction were recorded on shoot explants. whereas the ZE explants were the least

responsive. In addition. if no 2.4-D was included in the incubation medium for the whole

process. no SES were induced on cotyledonary or ZE explants. but 4 % of the shoot explants

gave an average of 2 SES per dish.

2. Effect of pretreatrnent

a) Stratified seeds

No interaction between GA, and light was found (Table 2.6). Growing the seedlings in light

or dark did not affect subsequent embryogenesis. The concentration of GA, influenced both

the fiequency and nurnber of somatic embryo (SE) significantly (Table 2.6, Fig. 2.10). The

highest frequency (55 %) and number (20) of SES produced per dish occurred in explants

grown on basal medium with no addition of GA,, while the fiequency and number dropped

to 40 % and 10 respectively when 1 pM GA, was added to the medium, and 18 % and 2 SES

with 5 or 10 pM GA, .

b) Green seeds

Light as a pretreatment was superior to darkness in both the fiequency of embryogenesis and

the number of SE per dish (Table 2.6). Early and mature stages of SES and multi-

cotyledonary SES were observed (Fig. 2.1 1). Some 52 % of the explants obtained fiom light

grown seedling gave rise to SES compared to 4 1 % from dark grown seedlings. The number

of SE per dish were 17 and 1 1 with light- and dark-grown explants. respectively. In addition,

the concentration of GA, af'fected both the fiequency and number of SES. Explants grown

on basal or 1 pM GA3 containing medium gave a similar frequency (5 1 - 56 %), whereas

explants obrained from 5 or 10 pM GA, containing medium gave a reduced fiequency of 36

- 43 %. The number of SES per dish decreased with increasing GA, concentration.

In these expenments, response to light and dark, and to the concentration of GA,,

were different in stratified and green seeds. Light did not affect embryogenesis in stratified

seed, but increased it in the case of green seed. On the other hand, the concentration of GA,

had a stronger influence on stratified seed than with green seed. In both cases, basal medium

alone or with 1 pM GA, (in case of green seed) were optimal as pretreatment for obtaining

seedlings for subsequent somatic embryogenesis using cotyledons as explants. Furthemore.

similar or higher frequency of embryogenesis were obtained from green seeds in cornparison

to stratified seeds (Table 2.6).

3. Effect of sucrose

a) Response of ZE explants to BM containing various levels (0-12 %) of sucrose

Cultures maintained on medium with various concentrations of sucrose for 8 weekî

In explants cultured on medium without sucrose, only 20 % (17 / 80) of the embryos

swived fkom contamination. and 4 of them gemùnated and grew to 15 mm long. No callus

or SES were found. On 1.5 - 12 % sucrose containing medium, only one or two embryos in

each treatment germinated. and seedlings were up to 18 mm long. Cotyledons, particularly

the ones in contact with the medium, offen enlarged up to 14 mm long (Fig. 2.12A). About

50 % of the embryos fomed small callus, mostly on the cotyledons. Occasionally. a few

adventitious bud-like structures were observed on cotyledons of embryos cultured on 3 - 9

% sucrose supplemented medium, while 20 % of the embryos cultured on 12 % sucrose

containing medium formed adventitious bud-like structures.

Pretreatment of cultures with various concentrations of sucrose for 2 weeks before

tramferring on to 3 % sucrose containing medium

Of 14 embryos which were cultured on 1.5 % sucrose for 2 weeks and survived from

contamination, one germinated with a seedling length of 20 mm. About 40 % of the embryos

formed callus on the cotyledons (Fig. 2.12B). Adventitious bud-like structures (up to 12)

were found on cotyledons of 2 embryos. On 6 - 12 % sucrose containing medium. 28 % ( 12

/ 43) of the embryos induced callus, and 3 - 12 adventitious bud-like structures were found

on 14 % (6 143) of the embryos.

b) Response of shoot explants to various amounts of sucrose (1 -5- 9 %) supplernented BM

Culiiires on medium with varioirs concentrations of sucrose for 8 wee ks

Browning was observed in most explants cultured in darkness on al1 concentrations of

sucrose tested (Fig. 2.12C). Small callus and bud-like structures were occasionally observed.

and 6 SES were found on one explant cultured on 9 % sucrose containing medium.

Most explants cultured under light were dark brown in colour, regardless of

concentrations of sucrose used. Occasionally. adventitious roots, which were brown in

colour as well as compact callus were observed but no SES were seen.

Pretreoimeni of cultures with various concenirations of sucrose for 2 weeks before

tramferring on to 3 % sltcrose containing medium

Most of explants cultured in darkness turned light brown with no callus. One explant

cultured on 1.5 % sucrose formed 2 adventitious bud-like structures. and two explants

cultured on 9 % sucrose containing medium formed 4 SES in total (Fig. 2.12D).

Almon al1 explants culhired under light in al1 concentrations of sucrose were dark-

brown in colour. Adventitious roots and small callus were occasionally found. and two

explants cultured on 9 % sucrose containing medium formed 4 SES.

Cornparhg the two types of explants, ZEs tended to gerrninate, some formed cailus

and adventitious bud-like structures, and 12 % sucrose seems to stimulate the formation of

bud-like structures. Browning was found in most of shoot explants regardless of the

concentration of sucrose in the medium; occasiondly, callus and adventitious roots and SES

were found.

4. Histology and SEM observation

The histology study confirmed somatic embryogenesis in American ginseng. Well-organized

globular (Fig. 2 .13A), early cotyledonary (Fig. 2.1 3B) and cotyledonary (Fig. 2.132) SES

were observed. The meristematic region was characterized by small cells with dense

cytoplasm and highly stained nuclei, and the embryos were loosely attached to the esplant

and had no vascular comection with it. Also the early stages of SES were found with SEM

(Fig. 2.14 A, B). Observation by SEM also showed morphologically mature cotyledonary

stage SES had similar shapes as mature zygotic embryos but about 10-60 % smaller. Both

somatic and zygotic embryos had smooth cotyledonary surfaces (Fig. 2.14D. E). Poorly

developed, short, hsed or multiple cotyledonary SES were also observed (Fig. 2. MC).

2.4 Discussion

In the present study. in general, when equivalent concentrations of 2,4-D and dicamba were

compared, the former was markedly more effective, both for the fiequency of SE induction

and nurnber of SES produced. Higher concentration (1 5 FM) of NAA was more comparable

to lower concentlation (5 PM) of 2+D with respect to the responses observed. In other plant

species, different types of auxin are also not equal quantitatively or qualitatively in their

action, and the effectiveness of an auxin is species and tissue specific (Hana et al., 1989:

Gleddie et al.. 1983: Jia and Chua, 1992; Meijer and Brown, 1987).

Auxin concentration strongly afTected the fiequency and nurnber of SE induction. The

results here indicated. generally, the fiequency and nurnber of SE induction increased as the

concentration of NAA increased from 5 to 15 pM in al1 three types of explants. With 2.4-D

and dicarnba, the highest fkequency and nurnber of SE were induced with 5 PM. Baker and

Wetzstein (1 994) reported somatic embryogenesis in peanut with 2,4-D or NAA. and showed

that as auxin level increased, Frequency of embryogenesis decreased. Reduction in

embryogenic potential by high 2,4-D levels has also been reported for papaya (Fitch and

Manshadt, 1990). On the other hand, fiequency of embryogenesis in creeping bentgrass was

increased fiom 57% to 7 1 % when the concentration of dicamba increased fiom 5 FM to 10

FM (Zhong et al., 199 1 ).

The influence of awin type on embryo morphology has been reported in rnany plants

(Banvale et al., 1986; Hartweck et al., 1988; Tabei et al., 1990). In the present study. some

SES obtained with NAA were of good quality - large, cotyledonary stage, and separate fiom

each other? but others were often fused together and to the parental explant tissue. Good

quality SES were also induced with 2,4-D and dicamba particularly with 5 pM of them. At

higher concentrations, the SES remained small and at the globular stage. In addition . cup

shaped SES were ofien observed with 15 p M dicamba.

Among the three types of explants tested, generally, shoots gave the highest

frequency and number of SES. the cotyledons were next. and the intact ZE the least

responsive. However, the variation was large, particularly with the number of SES produced

on shoot explants (Table 2.2 and 2.5). There are two possible reasons for this. Firstly. the

difference may be related to the portion of the "shoots" cultured. Some ''hoots" included

enlarged cotyledons. and cotyledons were more responsive than the shoots. Secondly, data

in Table 2.5 are fiom shoot explants subcultured every two weeks instead of four weeks. and

the fiesh medium may enhance the SE production. We noticed that 98.5 % of the SES

induced £rom seedling segments were formed on cotyledons. The origin of the explant and

its physiology are known to be very important factors determine the success of

morphogenesis (Ammirato 1983), and cotyledons have been s h o w the most responsive to

inductive medium for SE production (M-wthy et al., 1995, 1996; Tepper and Mante. 1990).

In the case of the combination of 2,4-D and NAA, the main effects of 2,4-D and NAA

have been considered since no interaction between 2.4-D and NAA was found. In generai,

similar to when 2,443 was used as a single growth regulator, higher frequency of SE

induction and number of SE induced were associated with lower concentrations of 2,443 in

the presence of NAA. In the case of NAA, concentrations tested did not show a significant

effect on embryogenesis in the presence of 2,4-D except with seedling segment explants in

which Iower concentration of NAA gave a higher fiequency. Both the frequency and number

of SES increased as the concentrations of NAA increased when N U was used as the only

growth regulator. This result indicates that 2,4-D exhibited a much stronger effect than NAA.

Exposure time to 2,4-D significantly intluenced the fiequency and SE production of

Arnerican ginseng in this study. Generally, exposure time of 4 or 6 weeks to 2,4-D (5 PM)

was optimal to promote hi& embryogenic potential. The influence of exposure rime in 2,4-D

containing medium on somatic embryogenesis has also been significant in some other crops

(Cook and Brown, 1995; Marsolais et al., 1991) . Finstad et al., (1 993) showed that there was

a consistent minimum requirement of exposure to growth regulators for embryogenesis to

occur in alfalfa petiole culture. The requirement for 2.4-D for initiation of SES in Amencan

ginseng has been reported by Li and Guo (1990), whereas SE production c m occur on basal

medium without growth regulators in Oriental ginseng (Choi et al., 1996; Lee et al.. 1990).

The present study indicated that somatic embryogenesis of American ginseng can occur on

basal medium but it is necessary for explants to have 4-6 weeks of exposure to 2,4-D

supplemented medium first as the inductive stimuli; the result was comparable to, or even

better than. those obtained fiom explants which remained on 2,4-D containing medium for

the whole period of induction. In to carrot somatic embryogenesis. auxin (2,4-D) was

required as the inductive stimuli but auxin removal was necessary for embryo development

(Li, 1992). The occurrence of SE with shoot explants on basal medium may be a result of the

carry-over effect of the explant since they were derived fiorn germinating SES.

The effect of pretreatment with GA, and light, for seedling growth and subsequent

embryogenesis using seedling cotyledonary explants was assessed. The addition of GA,,

particularly at higher Ievels (5 or 10 PM) inhibited subsequent embryogenesis on seedling

cotyledonary explants for both stratified and green seed of Amencan ginseng. Hutchinson

et al., (1997) reported that the addition of GA, in the induction medium for geraniurn

significantly reduced SE number per explant, while stimulating effects of GA, were shown

in femel (Hunault and Maatar, 1995) and in papaya (Chen et ai., 1987).

Although, light as a pretreatment for seedling growth did not significantly affect the

subsequent embryogenesis in stratified seed. it exhibited a strong influence with green seed

compared to dark pretreatment. The different response obtained fiom stratified and green

seeds could be related to the seed source and the developmental stage of the seeds. caused

by the different stratification processes for the seeds (Hovins, 1996; Proctor and Louttit.

1995). In other crops. light was required for embryogenesis in garden leek (Wang et al..

1995) and it stimulated SE formation in rye (Vazquez et al., 199 1 ; Vazquez and Linacero.

1995). On the contrary. the process of embryogenesis was suppressed by Iight in Camellia

(Sam-Jose and Vieitez 1993), Cyclamen (Takamura et ai., 1999, Podophyihz (Arumugam

and Bhojwani 1 990) and rye (Lu et al., 1 984).

Based on these studies we found that green seed derived explants exhibited similar

or even higher potential for SE induction than explants obtained from stratified seeds. This

finding implies that we could Save 7 months of stratification with green seed derived

explants.

In ZE explants, SES were not produced on MS basal media with various levels of

sucrose for various times, whereas SES were occasionally observed on MS basal medium

containing high level of sucrose in shoot explants. The influence of concentration of sucrose

on somatic embryogenesis has been shown in some other plants. Gray et al., (1993) reported

that 3 % sucrose stimulated higher percentage of explants producing SES than 1.5 or 6 %

sucrose, even though more SES per explant were produced on medium containing 2 %

sucrose. In soybeans (Lazzeri et al., 1987b), embryogenesis efficiency generdly declined

with increasing sugar concentration.

Summary

Somatic embryos (SES) of Amencan ginseng were induced on auxin supplemented MS

medium from zygotic embryos, seedling cotyledons and other segments, and shoots

obtained fiom SES. The SES occurred after 4 weeks of induction, and up to 68 % of the

explants produced SES afler 8 weeks of induction. In an experiment with al1 segments of

seedlings, over 95 % of the SES were induced from cotyledonary segments. Explants

obtained from green seeds exhibited similar or higher embryogenic fiequency as those by

explants denved from stratified seeds.

The key role of exogenous auxin(s) in controlling somatic embryogenesis was shown

in this study. Somatic embryos were not produced on MS basal medium without auxin(s).

Explants needed a minimum 4 weeks of exposure time to 2,4-D to induce higher quality SES.

Furthemore. the process of embryogenesis was regulated by auxin type and concentration

in the induction medium. The response to auxin was also dependent on explant types. The

auxins 2.4-D and NAA were effective for SE induction, although their optimal concentration

were different, and dicamba was Iess effective. In addition, as the concentration of NAA

increased from 5 to 15 FM, the fiequency and number of SE increased. whereas 5 pM 2.4-D

or dicamba gave beîter results than higher concentrations. When using combinations of 2,4-

D and NAA in the induction medium, the concentration of 2.4-D strongly influenced the

fkequency of embryogenesis.

The effects of pretreatment - GA, and light - for seedling growth of both stratified

and green seeds on subsequent somatic embryogenesis with seedling cotyledonary explants

were investigated. Light as a pretreatment stimulated the frequency of embryogenesis and

the number of SES from green seeds but did not influence the embryogenesis in stratified

seeds. The addition of GA, (1-10 FM) to zygotic embryo germination medium reduced the

embryogenic potentid in cotyledonary explants cultured on 2.4-D (5 PM) and NAA ( 1 5 PM)

enriched medium.

Table 2.1 Effect of 2.4-D, NAA, and dicamba on somatic embryogenesis in cotyledonary

and zygotic embryo explants d e r 8 weeks of induction.

Treatment Frequency # SE per dish (PM) (%)

Cotyledon Zygotic Cotyledon Zygotic embryo embryo

Control '

2,4-D ( 1 )

2,4-D (5)

2,4-D ( 1 0)

2,4-D (1 5 )

Trend y

NAA (5)

NAA (10)

NAA (15)

Trend

dicamba (5)

dicamba ( 10)

dicamba (1 5 )

Trend

' MS media with no growth regulator.

Significant (* ) at p=O.O5, L=linear, Q=quadratic.

Table 2.2 Effect of 2,4-D, NAA, and dicamba on somatic embryogenesis

in shoot explant (obtained form somatic embryos) of Arnerican ginseng

after 8 weeks of induction.

Treatment Frequency # SE (PM) (%) per dish

Control '

2,4-D (1)

2,4-D (2)

2,443 ( 5 )

2,4-D (8)

Trend y

( 5 )

NAA (IO)

NAA (15)

Trend

dicamba (5)

dicamba ( 10)

dicamba ( 15)

Trend

' MS media with no growth regulator.

y Significant (*) p=0.05, L=linear, Q=quadratic.

Table 2.3 Main effect of 2,4-D and NAA on soniatic eiiibryogenesis in cotyledoiiary, secdling segments, zygotic eiiibryo, and shoot (obtained froiii somatic eiiibryos)

explants afler 8 weeks of induction.

Treatment Frequency (%) # SE pcr dish (PM)

Coty ledon Seedling Zygot ic Shoot Cotyledon Seedling Zygotic Shoot segments ' eni bryo segnieiits em bryo

2,4-D 5 58.2 * 4.0 27.7 * 3.0 1 1.71 3.1 62.2 * 3.4 7.8 rt 0.6 6.4 * 0. I 0.5 * 2.0 15.5 * 0.9

Trend " L* L* L* L* L* L*

N A A 5 4 1.2 * 4.5 22.8 * 3.9 8.3 I 2.8 39.0 * 3.6 3.7 * 0.5 6.4 * O. 1 0.3 * O. 1 4.9 ~t 0.6

Trend L*

NS

NAA NS NJ NS NS NS NS NS

2,4-D x NAA NS NS NS NS NS N S NS Pi S

' Al1 segments of seedling.

Y The concentrations of 2,4-D used for shoot explant wcrc 3, 5, and 8 pM.

" Significant (*) nt p=O.O5, L=linear, Q=quadratic.

, * Nonsipificnnt or signifkant at p-0.05, respectivcly.

Table 2 . 4 Distribution of somatic embryos induced from seedling segments

after 8 weeks of induction on 3 concentrations of 2.4-D.

Treatrnent Distribution (%) 2,4-D (PM) Shoot Cotyledons Root

5 0.0 100 4.3 0.0

10 1.7* 1.7 95.5 * 5.3 2.8 i 2.8

15 0.0 100 * 5.2 0.0

Average 0.6 98.5 0.9 - - --

' The medium for somatic embryo induction contained 2,4-D and NAA.

the main effect of 2.4-D is presented.

Table 2.5 EEect of exposure time to 5 FM 2,4-D on somatic embryogenesis in cotyledonary,

zygotic embryo, and shoot (obtained fiom somatic ernbryos) explants afier 8 weeks of

Treatment Frequency (%) # SE per dish

(Weeks) CotyIedon Zygo t ic Shoot Coty Iedon Zygotic Shoot em bryo em bryo

O 0.0 0.0 4.2 k 2.8 0.0 0.0 0.3 * 0.7

2 34.2 k 6.1 2.6 i 1.8 62.5 * 9.0 3 .4I0 .8 0.1iO.O 21.3k3.2

4 47.2 k 7.8 10.5 St 5.5 68.8 * 8.2 6.2 k 1.2 0.4 * 0.7 42.3 k 6.1

6 44.4 k 7.2 15.8 * 4.4 66.7 * 7.7 4.4 1 .O 1 .O * 0.7 39.5 4.7

8 34.2 * 6.1 13.2 * 2.4 52.1 k 8.4 3 -6 * 0.8 0.5 * 0.6 30.3 * 4.4

Trend y Q* L* Q* Q* Q*

Z Explants were cultured on MS medium containing 2,4-D for various times and then

2.4-D was removed.

Significant (*) at p=0.05, L=linear, Q=quadratic.

Table 2.6 Main effect of light and GA, as pre-treatrnent on growth of seedlings fiom

stratified and green seeds on subsequent somatic embryogenesis in cotyledonary explants of

Amencan ginseng after 8 weeks of induction.'

P re-treatment Frequency (%) # SE per dish

Stratified Green Stratified Green seed seed seed seed

Light 32.3 * 3.3 52.1 I: 2.7 6.6 0.5 16.9 h 0.5

Dark 33.6h2.7 41.2h2.1 5.0 A 0.6 1 1 .S * 0.6

GA, (PM)

O 54.9 * 4.0 55.9 3.5 19.5 * 1 . 1 19.9 0.7

1 40.0*3.9 50.7k3.1 10.3 * 0.9 18.4 k 0.7

5 18.2h3.4 43.1*3.4 2.0*1.5 11.8*0.7

1 O 18.9 * 2.4 36.3 k 3.3 2.4 * 0.5 8.3 * 0.7

Trend y Q* L* Q* L* NS

Light

GA, I

Light x GA; NS

' Culture medium for SE induction contained 5 pM 2.4-D and 15 pM NAA.

Significant (*) at p=0.05, L=linear. Q=quadratic.

Ns , * Nonsignificant or significant at p=0.05, respectively.

Figure 2.1 Seed, zygotic embryo, and seedlings of Amencan ginseng.

A. Seed with cracked seed coat, and exposed endosperm (10.29,

B. Decoated seed, showing mature embryo (arrow) with endosperm

(1 0.29.

C. One week old seedlings obtained from zygotic emblyos which

germinated in vitro in darkness (etiolated seedling. right) or under

1 6h-photoperiod (green seedling; 2.8 x ).

D. Decoated seed, showing immature embryo (arrow) with endosperm

( 10.2~).

Figure 2.2 Cotyledonary explants cultured on 5 pM 2.4-D and 15 pM NAA

supplemented medium for 2 weeks. The explants were obtained fiom seedlings

cultured on:

A. MS basal medium (BM), under light (6.8x),

B. BM, in darkness (6.89,

C . BM supplemented with 10 p M GA,, in the light, showing elongation

and curling ( 6 . 8 ~ ) .

Figure 2.3 Cotyledonary explants responded differently with different auxins.

A.On 5 pM NAA containing medium, somatic embryos (SES) attached

to callus were induced, and adventitious roots were found (6 .89,

B. On 15 pM dicamba supplemented medium, a few cup-shaped SES

(arrow) were found (6.89.

Figure 2.4 Zygotic embryo explants after 5 weeks of induction.

A. Seedling grown on MS basai medium without any

growth regulator (4.59,

B. Adventitious roots developed with NAA ( 1 0 FM) enriched

medium ( 4 . 3 9

C . Callus was induced on 2,4-D (5 PM) supplemented medium

(4.3 x),

D. Seedling with elongated shoot on dicamba ( 5 PM) containing

medium ( 2 . 8 ~ ) .

Figure 2.5 Shoot explants afier 8 weeks of induction.

A. Brown explant cultured on MS basai medium without any growth

regulator ( 8 . 9 9 , showing no callus or SE.

B. Somatic ernbryos (SES) induced on 2,4-D (1 pM) containing medium

( 6 . 4 ~ ) .

C. SES induced on 2,4-D (8 PM) containing medium (6.4x),

D. SES and adventitious roots induced on NAA (IO pM) containing

medium (6.3 x ) .

Figure 2.6 Effect of a combination of 2.4-D and NAA on somatic embryo (SE)

induction with cotyledonary (A. B) and zygotic embryo ( C) explants.

A. SES induced on 10 p M 2,4-D and 5 pM NAA containing medium

(13.6~).

B. SES induced on 15 p M 2,4-D and 15 pM NAA containing medium.

showing small and globular stage SES ( 2 3 . 8 ~ ) ,

C. SES with multiple cotyledons were obsewed on 5 pM 2.4-D and

15 pM NAA containing medium ( 6 . 8 9

Figure 2.7 Effect of a combination of 2,4-D and NAA on somatic embryo (SE)

induction with shoot explants after 7 weeks of induction.

A. SES produced on 5 pM 2,4-D and 10 p M NAA containing medium

(1 0.29,

B. SES produced on 8 pM 2,4-D and 10 pM NAA containing medium,

staying in early developmental stage (1 0.29.

Figure 2.8 Somatic embryo (SE) induction in cotyledonary explants cultured on

5 pM 2,4-D supplemented MS medium for various times.

A. Brown explant which had no exposure to 2,4-D (6 .89,

B. Flower-like SES occurred widi explants had 2 weeks of exposure to

2,4-D ( 6 . 8 ~ ) ,

C. SES produced on explants had 8 weeks of exposure to 2,4-D (6.8 x ) .

Figure 2.9 Somatic embryo (SE) induction in shoot explants cultured on

5 pM 2,4-D supplemented medium for various times.

A. A few SES and adventitious roots occurred on explants which had

no exposure to 2,4-D (4 .39.

B. SES produced on explant had 4 weeks of exposure to 1.443 (4.39,

C. SES produced on explant had 8 weeks of exposure to 2.4-D ( 1 2 . 8 ~ ) .

Figure 2.10 Cotyledonary explants cultured on 5 pM 2.4-D and 15 pM NAA

supplemented medium for 8 weeks. The explants were obtained from seedlings

grown under light on:

A. MS basal medium (BM), showing many somatic embryos (SES) (6.8 x).

B. BM plus 5 p M GA,. showing only a few SES (arrow; 6 . 8 ~ ; ) .

Figure 2.1 1 Somatic embryogenesis in green seeds with cotyledonary explants on

2,4-D (5 FM) and NAA ( 1 5 FM) supplemented MS medium.

A. After 4 weeks of induction, showing early stages of somatic embryos

(SES, 2 7 2 ) .

B. Afier 8 weeks of induction, showing mature SES with 2 to 4 cotyledons

(27 .2~ ) .

Figure 2.12 Responses of zygotic embryo (ZE; A, B) and shoot (C' D) explants to

MS basal media without growth regdators but containing different levels of sucrose

for different times.

A. ZE cultured on 1.5 % sucrose containing medium for 8 weeks, showing

one enlarged and one small cotyledon, but no callus or somatic embryos

(SES, 6.4x),

B. ZE cultured on 12 % sucrose for 2 weeks then 3 % sucrose for 6 weeks,

showing calli around the edge of the cotyledons, but no SES ( 6 . 4 ~ ) .

C. Shoot explant cultured on 1.5 % sucrose containing medium for

8 weeks, with no callus or SE occurring ( 8 3 ) .

D. Shoot explant cultured on 9 % sucrose containing medium for 2 weeks

before transferred to 3 % sucrose containing medium for 6 weeks, showing

one SE ( 1 7 ~ ) .

Figure 2.13 Histology study of somatic embryogenesis.

A. Longitudinal section of cotyledonary explant d e r 8 weeks of induction

on 2,4-D and NAA containing medium, showing a globular snmatic embryo

(SE)'

B. Longitudinal section of cotyledonary explant d e r 8 weeks of induction

on 2,4-D and NAA containing medium, showing a early cotyledons SE.

C. Longitudinal section of cotyledonary explant after 8 weeks of induction

on 2,4-D and NAA containing medium, showing a cotyledons SE.

Figure 2.14 SEM observation of sornatic embryogenesis after 8 weeks of induction

on 2,443 containing medium (A-D) and a zygotic embryo (ZE, E).

Bar=0.1 mm in Aand 1 mm in B - E.

A. An early heart shaped somatic embryo (SE),

B. A early cotyledon SE.

C. A few abnormal SES, with multi cotyledons,

D. A mature SE. with two cotyledons and srnooth surface.

E. A mature ZE. having cwo cotyledons and smooth surface.

Chapter 3 Induction of somatic embryos - Effect of cytokinin in auxin

supplemented medium

3.1 Introduction

Combinations of auxins and cytokinins ha ive be en used for induction of somatic embryos

(SES) in many plants (Ahmed et al., 1994; Kiss et al., 1992; May and Trigiano, 199 1 : Mo and

Arnold. 1991; Rout et ai., 1991, Santos et al., 1994; Stolarz et al.. 1991 ; Tulecke and

Mcgranahan, 1985). The addition of cytokinins to the induction medium enhanced

embryogenesis in cantaloupe (Gray et al., 1993). creeping bentgrass (Zhong et al.. 199 1).

alfalfa (Finstad et al.. 1993), cyclamen (Takamura et al., 1995). and Acacia (Rout et al..

1995). Rugini (1988) reported that in olive, 75 day-old zygotic embryo (ZE) explants

produced SES on basal medium and addition of 0.5 or 2.5 FM benzylarninopurine (BAP) into

the medium enhanced the fiequency of SE induction; however, addition of NAA or 2.4-D

reduced or totally suppressed the potency of embryogenesis.

In pepper, SES were produced in the presence of both 2,4-D and TDZ, but not with

2,4-D alone (Binzel et al.. 1996). Similarly, SE production in Cayratia japonica was initiated

on 2,4-D combined with TDZ or kinetin (Zhou et al., 1994), while 2,4-D alone did not

produce any SES. Hatanaka et al., (1991) reported that leaf cultures of Coflea required

cytokinin (IP, kinetin, or BAP) as a sole inducing growth regdator, while low concentration

of 5 x 10" pM auxin (2,443 or NAA) reduced embryogenesis. Similarly, in Abies spp.,

cytokinin was used as the sole plant growth regulator for SE induction with immature ZE

explants (Norgaard and Krogstmp, 199 1 ; Schuller et al., 1989).

On the other hand the inhibitory effect of cytokinin in the SE induction medium has

been reported in pea (Kysely and Jacobsen, IWO). soybean (Lippmann and Lippman, 1984),

and peanut (Eapen and George, 1993).

Various cytokinin(s) have different effects (Das et al., 1997: de Wit et al.. 1990;

Eapen and George, 1993; Li& 1988; Reynolds, 1986). and the concentration of cytokinin

and the auxin / cytokinin ratio are other important factors in controlling the response

(Lippman and Lippman. 1984; van der Saim et al., 1996). However SE formation in blue

spruce occurred from ZE explants with a wide range of auxin / cytokinin ratios (Afele et al.,

1 992).

Combinations of auxins and cytokinins have been used for induction of SES in

Oriental ginseng (Arya et al., 1993; Cell6rova et al., 1992; Choi et al., 1982: Lee et al., 1990;

Lee et al., 1995). In Arnencan ginseng, 2,4-D (9 PM) and BAP (2.2 PM) have been used for

SE induction from ZE explants (Li and Guo. 1990); dicarnba (9 FM) and kinetin ( 5 PM) for

epicotyl and root explants (Tirajoh and Punja, 1995); and 2.4-D (9 PM) and kinetin (4.6 FM)

were used for root callus induction, followed by culture on dicamba (9 FM) supplemented

medium for SE formation (Wang 1990). In American ginseng, however. no comparative

study on the effect of the medium containing a u . alone and auxin combined with cytokinin

on SE induction has been reported. The effect of concentration of cytokinin in auxin

supplemented medium on somatic embryogenesis also remains unknown.

Therefore, the objectives of the midy were:

1 ) to investigate the effects of cytokinins (kinetin or BAP) in auxin (2,4-Do NAA, or

dicarnba) supplemented medium on SE induction in Arnencan ginseng;

2) to compare the effects of the two cytokinins, kinetin and BAP. and their concentrations

on embryogenesis in American ginseng;

3) to compare the responses obtained from cotyledonary and shoot explants to combinations

of auxin and cytokinin supplemented media.

3.2 Material and methods

Seed source

The seed source and seed stenlization were as descnbed in Chapter 2, but only stratified

seeds were used.

Explant v p e

1) Cotyledonary explants - Explants were isolated cotyledons of in vitro grown green

seedlings (see qgotic ernbryo germination in Chapter 2) .

2) Shoot - Shoots denved fiom SES were used as explants (see the flow chart in Fig.4.1).

Callus and somatic ernbryo induction

Induction medium consisted of MS basal medium (BM). B, vitamins. and one of three

aUXIns: 2,4-D (5 PM). NAA (IO PM). or dicarnba (10 PM). Kinetin or BAP at O. 0.1, 0.5. 1.

or 5 pM were also added to the induction medium. The pH of the media was adjusted to 5.6

before autoclaving. Al1 the cultures were incubated in a growth room at 24 O C in darkness.

Da fa collection and analysis

In most experiments, four explants were placed in one Petri dish with ten dishes (replicates)

for each treatment, and each experiment was repeated twice.

Cultures were exarnined under a dissecting microscope bi-weekly. As described in

Chapter 2, the time required for SE induction, embryogenic fiequency and the number of SES

per Petri dish were recorded at the 8th week of induction. and presented as mean * standard

error. A chi-square test was used for trend analysis o f the embryogenic frequency results

induced with different levels of cytokinins. Log transformation was carried out for the data

on number of SES, then the analysis of variance was performed using the General Linear

Mode1 (GLM) procedure of the Statistical Andysis System (SAS Institute, 1985), and back

transformed data are presented.

3.3 Resutts

1. Effect of cytokinin(s) in 5 pM 2,4-D containing medium on somatic ernbryogenesis in

American ginseng

Seedhg cotyledonary explants

Afier 4 weeks of induction, SES formed on explants cultured on 2,4-D alone or combined

with low concentrations of kinetin or BAP, but not on the explants cultured on 5 p M

cytokinins until the 8th week of induction.

m e n no cytokinin was added to the medium. SES were induced in 55 % of the

explants afier 8 weeks of induction (Table 3.1). Thirty six to 43 % of the explants cultured

on 0.1 - 1 pM kinetin containing medium, and 24 - 50 % of the explants on 0.1 - 1 pM BAP

containing medium produced SES. O d y 8 % or 5 % of the explants cultured on 5 pM

kinetin or BAP produced SES, respectively. Figure 3. l shows the SE production on medium

with or without cytokinin.

Shoot explants

SE formation was observed in al1 treatments at the 4th week of induction except 5 FM BAP

which did not induce SES until the 6th week. At the 8th week of induction. ernbryogenic

frequency (56 %) induced by 2,4-D (Table 3.1) was not afEected by 0.1 or 0.5 pM cytokinins

but reduced by 1 or 5 pM cytokinins. The number of SE per dish was reduced by addition

of cyto kinin(s).

In the absence of cytokinins in the medium, cotyiedonary and shoot explants

exhibited sirnilar frequency of embryogenesis. However. lower nurnber of SE per dish (8)

were induced from cotyledonary explants than from shoot explants (21). High levels of

cytokinin exhibited sboonger inhibitory effect with cotyledonary explants than with shoot

explants. For example, no SES were observed with cotyledonary explants cultured on 5 pM

cytokinin(s) until the 8th week of culture, whereas SE formation on 5 pM kinetin or BAP

containing medium occumed at the 4th or 6th week, respectively, in shoot explants.

2. Effect of cytokinin(s) in 10 pM NAA containing medium on somatic embryogenesis with

shoot explants

At the 8th week of induction. NAA alone resulted in a 64 % fiequency of SE induction

with 12 SES per dish (Table 3.2). When cytokinins were included with NAA, the frequency

and the nurnber of SES were both reduced significantly (Table 3.2).

3. Effect of cyîokinin(s) in 10 pM dicamba containing medium on somatic embryogenesis

Seedling coryledonary explants

SES were induced after 6 weeks of induction on dicamba alone or combined with low (0.1

or 0.5 FM) levels of cytokinin(s). Explants cultured on 5 pM kinetin or BAP containing

medium did not produce SES until the end of the experimental period (12th week of

induction).

At the 8th week of induction, 22 % of the explants induced SES on dicamba alone

containing medium (Table 3.3). Except for 0.1 pM BAP, addition of cytokinin(s) inhibited

somatic embryogenesis induced by dicamba.

77

Shoot explanrs

The 0.1 PM BAP supplemented medium stimulated the highest fiequency (33 %) among the

treatments at 8th week of induction (Table 3.3). compared to 15 % of the explants cultured

on dicarnba done. At 5 pM cytokinins reduced the fiequency to 8 %.

Dicamba alone at 10 pM induced low fiequency of SE induction in both co'tyledonary

and shoot explants. The fiequency was increased by adding low levels (0.1 or 0.5 PM) of

BAP with shoot explants.

3.4 Discussion

The critical role of auxins in SE induction of rimericm ginseng was s h o w in Chapter 2.

The study in this chapter reveals that addition of a cytokinin, kinetin or BAP, to auxin

supplemented medium had a significant influence on somatic embryogenesis, and the key

factor was the concentration of cytokinin(s). Generally, low concentrations of cytokinin had

no influence or a stimulating effect, but hi& levels of cytokinin suppressed the embryogenic

potency induced by auxin.

In Oriental ginseng, the eflectiveness of cytokinin(s) in auxin containing medium on

embryogenesis was also dependent on the levels of cytokinin(s) used. Kinetin (4.7 pM) in

4.5 FM 2,4-D containhg medium reduced the nurnber of secondary SES on primary SES in

Orientai ginseng (Arya et al.. 1993), but 2.3 pM kinetin was noneffective. Recently. Ahn et

al., (1996) reported a great stimulating effect on embryogenesis of Oriental ginseng by

adding a combination of BA and zeatin at 0.1 p M (BZ) to the 2,4-D supplemented medium

for seedling pretreatment and thin ce11 layer culture. In addition, as the concentration of BZ

increased. the embryogenic fiequency decreased but organogenesis increased. Similarly. the

inhibitory effect on embryogenesis of soybean in NAA (25 FM) containing medium was

shown at 0.23 pM BAP. but not at 0.045 FM BAP (Lazzeri et ai.. 1987a). In rose leaf culture

(de Wit et al., 1 WO), combinations of NAA (0.03 - 1.1 pM) with different cytokinins, BAP

(4.4 - 44.4 PM), kinetin (0.2 - 4.7 PM), and zeatin (4.6 - 9.2 PM) were tested, and SES were

only observed on the medium with NAA at 0.3 pM in combination with kinetin at 0.5 PM.

Reynolds (1 986) reported that Solanurn carolineme anthen cultured on 45.2 PM 2,4-D plus

4.7 pM kinetin supplemented medium for 4 - 8 weeks and then transferred ont0 a medium

lacking 2,4-D but supplemented with BA (2.2 - 44.4 PM) for SE induction. Low

concentration of BA promoted embryogenesis but inhibited organogenesis, and with

increasing concentrations of BA, regeneration was predominantly via organogenesis.

In the present study with shoot explants, d l the concentrations of BAP or kinetin

significantly reduced the frequency of SE induction and the nurnber of SE per dish induced

by IO p M NAA. High concentrations of kinetin or BAP reduced embryogenesis induced

in 5 PM 2.4-D or 10 FM dicarnba containing medium. In Lupinus, addition of a cytokinin

did not affect the fiequency of embryogenesis if 2,4-D was used. but lowered the fiequency

if NAA was the inducing auxin (Nadelska-Orczyk 1992). These results demonstrated that

the addition of cytokinin(s) to medium containing different auxins resulted in different

responses.

Explant type also influenced the response. In the present study with dicamba as the

inducing auxin, kinetin reduced the fiequency of SE induction with cotyledonary explants,

but did not affect the fiequency in shoot explants except at 5 PM. The requirement for auxin

and 1 or cytokinins, and their concentrations, for optimum production of SE dso differed for

various explant types in Cyclamen (Takamura et al.. 1995).

Kinetin and BAP did not exhibit significantly different effects in the present study.

In peanut (Eapen and George, 1993), both kinetin and BAP at 0.5 FM reduced the fiequency

of SE induction induced by 45.2 pM 2,4-D. Stronger inhibitory effect on embryogenesis of

BAP in cornparison to kinetin in 2,443 supplemented medium for embryogenesis has been

reported in soybean (Lippman and Lippman, 1 984). On the other hand, Litz ( 1 988) reported

that no callus initiation was observed on medium containing 2.4-D alone in longan leaf

culture, but callus was induced on most medium containing 2.4-D together with either BA

or kinetin, or BA alone. Furthemore, BA was much less effective for the induction of

embryogenic competency than kinetin, aithough it was more effective than kinetin for

stimulating callus formation-The mechanism of the effect induced by adding exogenous

cytokinin to embryo induction medium is not clear. Parrtt et al., (1995) suggested that the

role of exogenous cytokinin during the induction phase may depend on whether somatic

embryogenesis is direct or indirect. When embryogenesis is induced through the callus stage,

the fi-equency of SE formation is apparently enhanced by the presence of cytokinins in the

callus induction medium. In the direct system, on the other hand. where SES were produce

directly from explants. addition of cytokinin reduced the Sequency of SE formation. With

American ginseng as seen here, however, addition of two cytokinins generaily did not

stimulate the SE production, although the callus stage was involved in the process of the

embryogenesis.

The concentration of plant growth regulators in the medium, and a balance between

them. or auxin / cytokinin ratio. are important factors regulating regeneration (Das et al.,

1997; Hatchison et al., 1994). With Cyclamen seedling explants, 2,4-D at 0, 5, and 50 pM

was used in combination with kinetin at 0,0.5,5. and 50 FM (Takamura et al., 1995), and

much higher frequency of SE induction occurred in the medium with 2,4-D and kinetin at

10: 1 concentration ratio. On the other hand, different requirements for exogenous cytokinin

in different genotypes was s h o w in the adventitious root culture of rose (van der Salm et

al., 1996) where 2,4-D (O - 160 pM) combined with BAP (O - 50 PM) were used with two

cultivars for 8 weeks for callus formation, before the callus was transferred ont0 basal

medium. SE formation occurred in one cultivar in al1 treatments except for the calli induced

on medium with 50 p M BAP plus 2,4-D; in another cultivar, however. SE only formed on

calli induced on 50 pM 2,4-D oniy medium. In orchardgrass (Dactyiis giomeratu L., Wenck

et al., 1988), much higher levels of endogenous cytokinins were found in nonembryogenic

genotypes than a genotype showing embryogenic potential, and inhibition of embryogenesis

resulted fiom exogenously added zeatin. These results might be explained by genotypes

having different levels of endogenous auxin and cpokinins, and their ratio.

Generally. addition of cytokinins to awin containing medium did not stimulate but

supplernented somatic embryogenesis in American ginseng. The concentration of cytokinin

added to auxin containing medium was c~ucial for the response obtained. In general. adding

0.1 pM kinetin or BAP to auxin (2,4-D. NAA, or dicamba) supplemented medium, did not

affect the somatic embryogenic potency. Higher levels of kinetin or BAP. however. reduced

both the fiequency of SE induction and nurnber of SE produced. In addition. the types of

auxin in the medium, and the explant type used are other important factors controlling the

response. whereas the two types of cytokinins, kinetin and BAP, did not exhibit significantly

different effects in the present study.

Summary

Somatic embryogenesis of American ginseng was induced on MS medium supplemented

with auxin and cytokinh with green seedling cotyledons and shoots obtained fiom somatic

embryos (SES). The effects of hvo cytokiruns, kinetin and BAP with auxin (2,4-D, dicamba,

or NAA) supplemented medium were investigated. The process of embryogenesis was

delayed by inclusion of cytokinin in the induction medium, especially when higher

concentrations (1 or 5 FM) of cytokinins were used. Generally, kinetin and BAP exhibited

similar effects, and addition of cytokinin reduced somatic embryogenic fiequency and

number of SES produced per dish. However, stimulating effects of low concentration of

BAP (0.1 and 0.5 PM) were obtained with 10 FM dicamba containing medium in shoot

explants after 8 weeks of induction.

The Ievel of inhibitory eflect of cytokinin was explant specific. On 5 pM 2,4-D

containing medium. kinetin at 5 pM delayed embryogenesis until 8th week in cotyledonary

explants, but SES formed on 5 p M kinetin containing medium after 4 weeks of induction in

shoot explants.

This study showed again, that 2,4-D (5 PM) or NAA (10 FM) were more effective

than dicamba (10 PM) in inducing embryogenesis in Amencan ginseng. In addition. the

response to addition of cytokinin to the auxin containing medium was dependent on the

auxin type used. In cultures of shoot explants with 0.1 pM BAP, the fiequency of

embryogenesis was increased or decreased in 10 pM dicamba or 10 p M NAA e ~ c h e d

medium, respectively, and was not effective if 5 pM 2,4-D was used as the inducing auxin.

Table 3.1 The effect of cytokinins. kinetin and BAP, supplemented to 2.4-D (5 PM)

containing medium on somatic embryogenesis in cotyledonary and shoot explants d e r 8

weeks of induction.

TreatmenUpM) Frequency (%) Number of SES per dish

Kinetin BAP Cotyledons Shoot Cotyledons Shoot

O 55.3 * 5.3 55.6 * 4.5 8.8 * 1.0 31.1 * 1.5

O. 1 42.5 * 7.3 52.9 i 5.0 3.7 * 0.7 13.3 k 1.6

0.5 36.3 * 7.1 56.3 * 5.9 2.3 * 0.7 11.1* 1.4

1 .O 4 1.3 k 7.3 45.8 * 4.9 1.9 * 0.6 7.9 * 1 .O

5.0 7.5 * 3.7 34.0 * 4.2 0.3 k 0.4 3.1 0.5

Trend " Q* L* Q* Q* O 55.3 i 3.3 55.6 i 4.5 8.8 * 1 .O 31.1 2 1.5

0.1 50.0 5 7.3 59.7 * 4.8 6.8 * 1 .O 15.8 * 1.3

0.5 41.3 * 7.8 51.4 i 4.1 3.3 0.8 15.2 * 1.4

1 .O 23.8 * 4.2 31.0 * 4.7 1.8 * 0.6 5.7 * 0.8

5 .O 5.0 2.3 20.1 * 4.7 0.1 * 0.0 1.3 * 0.6

Trend Q* Q* Q* Q*

' Significant (*) at p=0.05, L=linear, Q=quadratic.

Table 3.2 The effect of cytokinin in N U (1 0 PM) containing medium

on somatic embryogenesis in shoot explants after 8 weeks of induction.

Treatrnent (FM) Frequenc y Number of (%) SE per dish

Kinetin BAP

0.5

1 .O

5.0

Trend '

0.0

Trend

Table 3.3 The effect of two cytokinins, kinetin and BAP, in dicarnba ( 1 0 FM) containine

medium on somatic embryogenesis in cotyledonary and shoot explants after 8 weeks of

induction.

Treatment (FM) Frequency (%) Number of SES per dish

Kinetin BAP Cotyledons Shoot Cotyledons Shoot

O

o. 1

0.5

1 .O

5 .O

Trend "

0.0

o. 1

0.5

1 .O

5.0

Trend

' Significant (*) at p=0.05, L=Iine- Q=quadratic.

Figure 3. l Effect of cytokinin in 2.4-D containing MS medium on somatic

embryogenesis with cotyledonary explants. Explants were cultured for 12

weeks on 5 pM 2,4-D containing medium supplemented with:

A. No cytokinin. showing rnany somatic embryos (SES) (6.8x),

B. 5 pM BAP, showing a few SES ( 6 . 8 9

Chapter 4 Conversion and development of Plantlets obtained from

somatic embryos of Arnerican ginseng

4.1 Introduction

"Seed germination is perhaps the most precarious time in the life of a plant" (Dure 1975).

With somatic embryos (SES). plant regeneration from them is also a key step. Plantlets were

readily obtained fiom SES of plant species such as Acacia (Rout et al., 1999, Cayrotiu

japonica (Zhou et al., 1994). Chinese cabbage (Choi et al., 1996), and weeping bamboo

(Woods et al.. 1992). However. in plant species such as Rosa hybrida L. cv. Landora (Rout

et al., 199 l), Vitis (Rajasekaran et al.. 1982). and walnut (Tulecke and Mcgranahan. 1985).

cold treatment was required to obtain normally developed plantlets by breaking the

dormancy in SES. In Nerium oleander, conversion of the primary SES into plantlets was

compromised by secondary embryogenesis (Santos et al., 1994). Low conversion rate in

horse chestnuts was due to asynchronous maturation and low germination (Capuana and

Debergh. 1997). The conversion rate fiom SES can be genotype specific. May and Trigiano

(1 99 1 ) reported that, among the 12 chrysanthemum cultivars which produced SES, plantlets

were only obtained From SES of 5 cultivars, and the conversion rate ranged fiom 0-23 %,

depending on the cultivar.

Many factors are known to influence this process, such as the composition of the

growth media for somatic embryo growth, maturation and germination. In lupin species,

development of plantlets obtained fiom SES needed an appropriate sequence of growth media

(Nadolska-Orczyk 1992), and abscisic acid (ABA) was the key element in the media. ABA

has long been considered as a regulator of seed germination (King 1982). ABA plays a role

in seed maturation, preventing precocious germination and inducing domancy. ABA

regulates development in SES of alfalfa (Lai and McKersie 1994), and plays an important

role in the maturation of conifer SES (Attree and Fowke, 1993).

In addition. carbohydrate influences SE morphology and conversion (Stnckland et

al., 1 987). Casein hydrolysate (CH) af5ected maturation of SES in white spmce (Barrett et al.,

1997), and was used in longan for SE maturation (Litz 1988). Gibberellins may be required

for seed development (Khan 1982), and have stimulated rooting of SES, and subsequent

growth of the plantlets (Amrnirato 1983).

In Oriental ginseng, a combination of 7.9 FM gibberellic acid (GA,) and 4.4 pM BAP

was used universally for plantlet regeneration from SES which were denved fiom zygotic

embryos (ZEs. Lee et al., 1990). cotyledonary explants (Lee et al., 1995), and protoplasts

cultures (Arya et al., 1993). However, only shoots were produced when the same

combination of growth regulators was used with the SES derived fiom root expiant (Chang

and Hshg, 1980). In addition. 1.45 pM GA, was used for plantlet formation From flower bud

derived SES (Kishira et al., 1992).

In Amencan ginseng, plantlets were obtained from SES produced from zygotic

embryos (ZEs, Li and Guo, 1990) and root explants (Wang 1990) in the presence of various

plant growth regulators, include GA,, BA, NAA, and 2,4-D, but the conversion rate was ~ O W

(30 %, Wang 1990). When 4.4 pM BAP and 2.9 pM GA, were included in the medium, only

shoots were regenerated from SES after 1.5 months of culture (Tirajoh and Punja, 1995).

The objectives of this study were:

1 ) to compare the conversion rate of SES into plantlets on !4 MS basal medium (% BM)

supplemented with or without GA, and BA;

2) to test the effect of sucrose, CH and ABA on plantlet growth:

3) to compare the germination of stratified seeds, ZEs, and SES on % BM alone or

supplemented widi GA,.

4.2 Materials and Methods

1. Influence of GA, and BAP on plantlet conversion and growth.

First generation somatic embryos (SES)

In one set of experiments, four-month-old SES obtained fiom stratified seed (Fig. 4.1 )

were used. Individuai SES were collected fiom the callus tissues under a dissecting

microscope and cultured in Petri dishes. Two types of media, viz.. a. half strength MS salts.

B5 vitamim. I .5 % sucrose, and 3 % gelrite (Yi BM) plus 1.45 p M GA,; b. !h BM plus 1.45

p M GA, and 2.2 pM BAP were tested.

In addition, four-month-old SES obtained from green seeds were used. The following

procedures were carried out: a. germinating zygotic embryos (ZEs) of green seeds on 1 or

3 pM GA, e ~ c h e d MS medium under dark or light (16 h - photoperiod per day); b. excising

the two-week-old seedlings into 5 mm long segments, then cultured on 5 pM 2.4-D and 15

FM NAA supplemented MS for 4 months; c. individual SES were collected and cultured on

% BM supplemented with 1.45 pM GA,.

The following categories were used for cornparison of the effect of growth media on

SE growth and recorded d e r 4 weeks of culture. 1 ) Conversion rate, which is the percentage

of SES developed into plantlets (having both shoots and roots) among the total number of

SES cultured; 2) Percentage of SES forrned shoots only, referred as '-shoots"; and 3)

Percentage of "other" SES, including small SES which did not develop M e r ; SES having

elongated radicle only; and green structures with no identified shoots or roots.

Plantlets developed from stratified seed- derived SES were transferred ont0 L/2 BM

after 4 weeks of culture. After 10 weeks of culture, the plantlets were removed fiom the

medium, rinsed out the agar with sterile distilled water, and planted onto sterile J i Q 7

pellets. The transplanted plantlets were placed in magenta culture vessels with cover.

watered with 114 MS salts, and kept in a growth chamber in 1 6h / 8h (light / dark) and 22 /

16 OC (day / night). After one week of transfemng, the covers of magenta boxes were

gradually opened and removed. But the boxes were kept in a green tray with clear plastic

cover. The plaiîtlets (with Ji@ pellets) were transferred into plastic pots with Promix-BX

afier about one moiith. and kept in the growth chamber under sarne light and temperature

condition. Growth pararneters including height of shoots and roots. number of shoots.

diameter of root and number of leafiets per shoot were recorded on 4-month-old plantlets.

Second generation SES

For secondary generation SES (see fiow chart in Fig. 4.1), four culture media: a. !4 BM

alone: b. % BM supplemented with 1.45 pM GA, ; c. !4 BM plus 1.45 piM GA, and 2.2 pM

BAP: and d. !4 BM containing 2.9 pM GA, and 4.4 pM BAP were used for 4 weeks, and

then the growth regulator(s) were removed.

Rates (%) of SE developed into plantlets. shoots and others (same as descnbed

previously) were recorded after 4 weeks of culture. Growth pararneters (same as descnbed

previously) and in vitro flowering were recorded after 1 0 weeks of culture.

2. Effect of sucrose, casein hydrolysate (CH), and abscisic acid (ABA) on plantlet growth.

92

Third generation SES (see flow chart in Fig. 4. l ) , which were derived from shoot explants

cdtured on 2.4-D (5 pM) for 6 weeks and then on MS basal medium for 4 weeks, were used

in these expenments. The culture medium consisted of half strength MS salts, B, vitamins,

and 3 % gelrite. Sucrose at 1 S. 3,6. or 9 % was added to the medium combined with or

without CH (5 g / L). In an another set of expenments, ABA at O, 1 . 2, 4, and 8 FM was

added to the same basal medium containing 1.5 % sucrose (!4 BM).

SES were incubated on the above media for two weeks, then transfemed ont0 !4 BM

supplemented with 1.45 pM GA, for 4 weeks. Growth parameters described previously were

recorded f i e r 6 weeks of culture.

3. Germination of seeds. zygotic embryos (ZEs) and SES.

Stratified seeds were sterilized with 40% bleach for 10 min, followed by 3-5's rinses of

sterile distilled water, then planted in Y' plastic pots (four seeds per pot) with Promix-BX.

The cultures were maintained in a greenhouse under 30 % shade.

The procedure for obtaining and sterilizing ZEs (3.7 mm long) From stratifîed seeds

was the sarne as that described in Chapter 2. Third generation SES (see flow chart in Fig. 4.1)

which were about 1.7 mm in length were also used. For both zygotic and somatic embryos,

ten Petri dishes were used with four embryos in each dish, and % BM with or without GA,

(1.45 FM) were used as growth medium. After 2 weeks of culture, GA, was removed from

the culture medium, and the cultures were kept for another 2 weeks. Germination rate (which

represents the percentage of seeds or embryos which had radicle of O S mm or longer) was

recorded at 2 and 4 weeks of culture.

93

In most experiments, four embryos were cultured in one Petri dish with twenty

dishes (replicates) for each treatment, and each experiment was repeated twice. Cultures were

kept under light (l6h-photopenod) at 24 O C . The andysis of variance was perfomed using

the General Linear Mode1 (GLM) procedure of the Statistical Analysis Systern (SAS

Institute, 1985).

1. Effect of GA, and BAP on plantlet conversion and growth

First generarion SES

SES produced from stratified seed derived seedling explants were used in th is expenment.

After 4 weeks culture on !4 BM supplemented with 1.45 pM GA,, about 85 % SES were

converted into plantlets which had well developed shoots and roots (Table 4.1 ). On medium

containing both GA, ( 1.45 PM) and BAP (2.2 PM), however, 58 % of the SES converted into

plantlets, and 25 % of the SES developed shoots only.

In an another set of experiments, SES produced from green seeds denved seedling

explants (with various pre-treatrnents) were used. The concentration of GA, (1 or 3 PM) as

pretreatment did not affect the conversion rate. However, SES produced from light-grown

seedling explants gave higher conversion rate (47 %, Table 4.2) compared to those produced

from dark-grown seedlings (3 1 %). Figure 4.2 shows a plantlet with two stems d e r 3 weeks

of culture.

Second generaiion SES

The highest conversion to plantlets occurred with SES on 1.45 pM GA, containing medium

(89 %, Table 4.3) after 4 weeks of culture. The conversion rate dropped to 44 % when 2.2

pM BAP was added to the medium, and 56 % SES converted to plantlets when 2.9 p M GA,

and 4.4 PM BAP were included in the medium. Only 16 % SES developed into plantlets,

while the most SES (76 %) remained small and did not develop M e r on % BM contained

no growth regulator. Figure 4.3 shows the plantlets obtained with al1 treatments.

95

Afier 10 weeks of culture, plantlets grown on different media exhibited similar

growth (Table 4.3). Plantlets with 3-4 shoots, and had less than 3 leaflets per shoot were

observed in al1 treatments.

2. Effect of sucrose, casein hydrolysate (CH), and abscisic acid (ABA) on plantiet growth

Third generation SES were used in the experirnent. SES were cultured for 2 weeks with 1.5 -

9 % sucrose and CH (O or 5 g/L), or sucrose (1.5 or 3 %) and ABA (0-8 PM), and then

transferred to 1.45 pM GA, supplemented % BM for 1 weeks. The 2 weeks exposure to

various levels of sucrose and CH affectecf the plantlet growth (Table 4.4). For example, the

plantlet height (shoot plus root) were 26 and 19 mm for the plantlets grown on CH free and

with CH in the medium. respectively (Table 4.4). Dry weight of the total plant material per

dish was higher with the plantlets grown on 1.5 % sucrose containing medium than those

grown on 3 -9 % sucrose containing medium. On the other hand, plantlet growth fiom SES

grown on various levels of ABA did not exhibit significant difference after 4 weeks culture

on GA, (1 -45 FM) containing medium except that plantlets grown on ABA free medium had

higher fiesh and dry weight than those grown on ABA containing medium (Table 4.4).

3. In vitro flowering of SE-derived plantlets

In vitro flowering was observed in plantlets converted from secondary generation of SES

grown on GA, (1.45 or 2.9 FM) and BAP (2.2 or 4.4 PM) enriched !h BM d e r 10 weeks of

culture. On 1.45 FM GA, containing medium, about 10 % of the plantlets formed flowers.

With plantlets grown on !4 BM enriched with other combination of GA, and BAP, in vitro

flowenng was occasionally observed. Compared to the flowers on the greenhouse-grown 3-

year-old plants (Figure 4.4). flowers of SE-derived plantlets were about 50 % smaller and

had significantly longer filaments. Single flowers were observed most of the tirne, although

inflorescences with up to 12 flowen were aiso observed (Figure 4.5 and 4.6). Flowen had

5 sepals and 5 petals (both were light-green in colour). and 5 yellow anthers (Figure 4.7 a).

However, flowers with less or more than 5 anthers were also observed (Figure 4.7 b). Pollen

grains were observed after staining d e r aceto-carmine (Figure 4.8). Under a scanning

electron microscope, anthers, pollen grains, and the grain surface were compared between

greenhouse-grown plant and SE-derived plantlet (Figure 4.9). Some pollen grains of SE-

denved plantlet were poorly developed and varied in size, although the pattern of the exine

sculpture was similar to those of greenhouse-grown seedling.

4. Plantlet transplantation

Four-week-old plantlets grown on GA3 containing medium were cultured on !4 BM for 6

weeks (Figure 4.10 a) and then transferred on Ji@ 7 pellets and Promix-BX. and kept in a

growth chamber (Figure 4.10 b). The measurement of 4 month-old plantlets showed that.

both shoot and root height were about 1w2.7 mm; shoot number was 2.8k 0.3: root diameter

2.9 kO.3 mm; and leaflet number per shoot was 1.8k 0.2. Figure 4.1 1 shows a 3 month-old

plantlet which had well developed shoots and tap roots.

The upper parts of the plantlets died back afier 4-5 months in the growth chamber,

but the roots were alive. After 3 months of storage at 3 OC, a few roots were still alive and

the bud started to emerge in the cold, and this process was similar to that occurred in

greenhouse-grown seedlings (Figure 4.1 2).

5. Cornparison of germination arnong seed, zygotic embryos (ZEs), and somatic embryos

( S W

Only 5 % seeds geminated in Prornix-BX after 4 weeks of culture (Table 4.5). No seeds

without seed coat germinated in vitro in this experiment, even when GA, was included in the

medium. Contamination was observed in the seed cultures. On the contrary, 85 - 87 % of the

dissected ZEs gemiinated on either !4 BM alone or containing GA, (1.45 PM) &er 2 weeks

of culture. The germination rate did not change but the seedling height increased d e r 4

weeks of culture.

After 2 weeks of culture, 32 % and 92 % of the SES germinated on '/z BM alone and

1.45 FM GA, containing medium. respectively. The rate increased to 47 % with basal

medium grown SES after 4 weeks of culture and remained the sarne with SES g r o m on GA,

containing medium. Figure 4.13 shows the germinating embryos.

4.4 Discussion

1. Effect of GA, and BAP on plantlet conversion and growth

In the present snidy with fim generation SES, about 85 % of the SES derived fiom stratified

seeds converted into plantlets which had both shoots and roots d e r 4 weeks culture on 1.45

pM GA, contained medium. On the contrary, 58 % of the SES converted into plantlets and

25 % of the SES developed shoots only when 2.2 pM BAP was also in the medium. It seems

that the presence of BAP inhibited root formation in some of the SES (Gill et al., 1995; Van

Schaik et al., 1996). Similarly, with second generation of SES, plantlet conversion was 88

% with SES on medium supplemented with 1.45 pM GA, and conversion rate reduced to

half when BAP was also in the medium, and 56 % of the SES converted into plantlets on 2.9

p M GA, and 4.4 pM BAP containing medium. Tirajoh and Punja (1995) reported that in the

presence of 2.9 pM GA, and 4.4 pM BAP, SES which were derived From root, leaf, and

epicotyl explants of Amencan ginseng did not develop roots after 1.5 months of culture. The

different results obtained with 3.9 PM GA, and 4.4 pM BAP containing medium could be

the result of different explant source, and the influence of the culture history (Ranch et al.,

1 985). In alfalfa, SES initiated on medium containing zeatin-riboside and 2.4-D usually

produced both shoot and roots, while SES cultured on BA and 2.4-D medium usually had

retarded primordial roots (Kao and Michayluk, 198 1).

A low conversion rate was obtained with '/z BM afler 4 weeks of culture in this study,

and addition of 1.45 pM GA, to the medium significantly improved conversion rate. Jia and

Chua (1992) reported that both the cytokinin and auxin were required for SE maturation and

germination in Pharbiris nil, and abnomally swollen structures instead of normal plantlets

were obtained fiom SES which were directly transferred to basai medium without growth

regdators.

in case of green seed derived SES, conversion rate was afTected by the pre-treatment

for seedling growth. Somatic embryo induction was hfiuenced by pre-treamient as discussed

earlier (Chapter 2). Light as pre-treatment was better than darkness not oniy for SE

production but also gave a higher conversion of SE to plantlets.

Ginseng plant usually has one solitary shoot. However, many of the plantlets

developed fiom SES had more than one shoots, often two or three. This could be related to

the environment of in vitro culture. and the effect of growth regulators in the growth

medium. Seedlings germinated fiom ZEs of Oriental ginseng also developed 2-3 shoots on

5 FM GA, containing medium (Lee et al.. 1 99 1 ).

2. Effect of sucrose, CH, and ABA on plantlet growth

In this study, 2 weeks of exposure to various concentration of sucrose, CH, and ABA had

influence on plantlet growth after 4 weeks culture on GA, containing medium. Longer shoots

and roots were observed in plantlets grown on a lower level of sucrose (1.5 %) than those

on higher levels, and plantlet height was reduced by adding CH (5 g/L) to the medium. In

addition, plantlet height and weight were reduced by addition ABA to the medium. In other

crops, ABA has been well docurnented for its role on SE maturation and plantlet conversion.

However, its effect was dependent on a couple of factors, including the method of

sterilization for ABA, the age of culture, and the growth regulators in the medium for SE

induction (Attrr and Fowke, 1 993; Lai and Mckersie, 1994; Senaratna et al., 1990).

3. In vitro flowering

Plantlets denved fiom secondary generation SES formed flowers on K BM supplemented

with GA, alone or combined with BAP afler 10 weeks of culture. Usually, American ginseng

plants need a 2-3 year juvenile penod to reach the flowering age in the field (Proctor and

Bailey, 1987). In vitro culture shortened flowenng penod to a few months (Fig. 4.1).

In vitro flowering has also been observed in Oriental ginseng with SES (Chang and

Hsing. 1980; Shoyama et al.. 1988; Lee et al., 1995), and ZEs (Lee et al., 199 1). BA but

not GA, was suggested to be required, although both of BA (4.4 FM) and GA, (1.45 or 2.9

PM) were included in the medium which induced in vitro flowering. it is well known that

cytokinin is a cornrnon requirement for in vitro flowering (Scorza, 1982), and combination

of the cytokinin with GA, could enhance the effect. On the other hand. gibberellins applied

in vivo promote flowering of some species and inhibited flowenng of others (Pharis. 1 985).

4. Germination of seed, zygotic embryos, and SES

Afier 4 weeks of culture. isolated ZEs germinated well in vitro on 55 BM alone or

supplemented with 1.45 pM GA,. On the contrary, seeds without seed coats did not

germinate, even when GA, was included in the medium, while Z seeds germinated in Prornix

in the presence of seed coat. Seeds coats and endosperm have endogenous ABA, which is

an extremely potent inhibitor of germination (Black 1972; Kelly et al.. 1992). Tirajoh and

Punja (1995) reported that 4 to 6 months was required for stratified seeds of American

ginseng without seed coats to germinate on GA, and BAP containing medium in cold

temperature. Seeds of Donglas-fir germinated poorly at 23 O C but isolated embryos

germinated hlly within 2 weeks, and this was correiated with a difference in ABA level

(Bianco et al., 1997).

In the present study, % BM alone or supplemented with GA, (1.45 PM) did not

change the germination rate in ZEs of stratified seeds, but made a significant difference in

the case of SES. These results indicate that, ZEs at a size of 3.7 mm (stage III. Proctor and

Louait, 1995) can germinate in absence of GA,, but SES need a supply of GA, in the

medium to obtain higher rate of germination.

Summary

Plant regeneration was obtained by gemiinating somatic embryos (SES). On half strength MS

basal medium without any growth regulators, 16 % SES developed into plantlets after 4

weeks of culture, while 1.45 p M GA, gave a conversion mte of over 80 %. On the contrary,

addition of BAP reduced the rate dramaticaily.

Two weeks exposure to various levels of sucrose , casein hydrolysate (CH). or

abscisic acid (ABA), intluenced plantlet height, and fiesh and dry weight of plant materials

4 weeks after SES were transferred to GA, containhg medium.

In vitro flowering was observed in 10 % secondary generation of SES derived

plantlets on 1.45 p M GA, conîaining medium, and occasionally on both GA qnd BAP

supplemented media. Single flowers, or inflorescences composed of up to 12 flowers were

found. Flowers were smaller in size but with longer filaments than those of greenhouse

grown plants. Flowers had five sepals and petals, one stigma, and five filaments, although

2, 3, or 8 filaments were also observed. Pollen grains were observed in anthers of those

flowers. Scanning electron microscope observation revealed that those grains had similar

exine sculpture as those obtained fiom greenhouse grown plants.

SE-denved plantlets were successfully transplanted to soilless mixtures. and

maintained in a growth chamber under controlled temperature and light condition.

Germination of seeds, zygotic embryos (ZEs), and SES on !4 MS basal medium

supplemented with or without GA, were compared. ZEs, but not seeds , germinated in vitro

on both media tested. Addition of GA, enhanced the germination in SES.

Table 4.1 Effect of GA, and BAP on rates of plantlet (conversion), shoot and other SES afier

4 weeks of culture ".

- - - -

Treatment Rate (%)

Basal GA3 BAP Plantlet Shoot O ther :' media (PM) (FM)

!4 BM 1.45 O 84.7 * 2.7 2.7 i 1.1 12.6 * 2.6

' First generation SES which were denved fiom stratified seeds were used.

y Those were: 1 ) small SES, did not develop M e r ; 2 ) roots developed, but no shoots

o c c m d ; 3) green structure, but no shoot or roots could be identified.

Table 4.2 Effect of pretreatment for seedling growth on rates of plantlet (conversion). shoot

and other SES d e r 6 weeks of culturez.

Pre-

Treatment

Rate (%)

Shoot Other y

Light 47.5 * 4.9 25.2 * 5.4 27.3 k 4.8

Dark 31.4 4.9 23.1 * 3.8 45.5 * 4.9

GA, (PM)

1 40.8 * 4.5 23.0 * 4.2 36.2 * 4.5

3 39.0 * 5.5 25.2 * 5.2 35.8 ~t 5.4

' First generation SES which were derived fiom green seeds were usai. Culture medium for

plantlet conversion consisted % BM and 1.45 pM GA,.

y Those were: 1) small SES, did not develop M e r ; 2) roots developed. but no shoots

occurred; 3 ) green srnichire. but no shoot or roots could be identified.

Table 4.3 Effect of GA, and BAP 011 rates of plantlet (conversioii), shoot and other SES (data collected afler 4 weeks of culture) and growtli

of plantlets after 10 weeks of culture '.

Treatrnent

Basal GA, BAP Plantlet Shoot 0th medium (PM) (PM)

Shoot Shoot Root Root Leaflet nuniber height lengtli diameter nunibcr

' Second generation SES were used.

Table 4.4 Effect of sucrose and casein hydrolysate (CH) (upper part of table), and sucrose

and ABA (lower part of table) on growth of plantlets after 6 weeks of culturez.

Treatment Shoot Shoot Root Root Leafiet Fresh Dry number height length diameter nurnber weight weight

(mm) (mm) (mm) (mg)' (mg).'

Sucrose (%)

1.5 1.6 10.2 14.0112 15.6 '1.3 1.4 +O. 1 1.6 I O . 1 0.1 1 I 0.05 O. 1 1 I 0.06

3 .O 2.6 20.5 12.2k1.2 12.821.0 1.3 IO. 1 1.3 I O . 1 0.51 I 0.06 0.05 rt 0.01

6.0 1.8 r0.1 9.8 I I . 3 10.9 k l .2 1.2 +O. 1 1.1 I O . 1 0.38 I 0.03 0.04 I 0.00

9.0 1.7 20.2 9.4 11.1 9.0 20.7 1.3 10.1 1.1 10.1 0.44 I 0.07 0.04 + 0.0 1

Trend ' L ' L t

CH @IL)

O 2.1 k0.2 13.0&0.8 13.820.9 1.4=0.1 1 .4 IO. 1 0.46 * 0.05 0.08 I 0.03

5 1.7 10.2 9.6 10.8 10.2 i0.6 1.2 10. f 1.1 20.1 0.4 1 + 0.03 0.04 i 0.00

Sucrose (%)

I .j

3 .O

ABA (PM)

O

1

1 - 4

8

"SES were cultured with various concentrations of sucrose and CH or ABA for 2 weeks,

and then on half-strength BM medium containing GA, (1.45 FM) for 4 weeks.

Fresh and dry weight per 4 plantlets in one dish.

' Significant (*) at p=0.05. L=linear.

Table 4.5 Cornparison of germination of seeds, zygotic embryos (ZEs) and somatic embryos

(SES) after 2 and 4 weeks.

Treaiment Germination (%) Height of seedling / plantlet

Matenal Media 2 weeks 4 weeks 2 weeks 4 weeks

Seed (with seed coat)

Seed (without seed coat)

Seed (without seed coat)

ZEs

% BM+ 1.45 FM GA, O O

55 BM+ 1.45 pM GA, 92.5 I 5.3 92.5 I 5.3 8.9 0.7 mm 22.1 I 3.5 mm

Figure 4.1 In vitro regeneration of American ginseng.

Stratified seeds

Zygotic embryos MS salts, 85 vitamins (W* + 1-2 weeks GA3 1 or 3 FM, darWlight

Seedlings BM, 2,4-D (5, 10, 15 FM), + 5-9 wee ks NAA (5, 10, 15 FM)

SES (1 st generation)

112 BM, BAP (2..2 FM), GA3 (1 -45 FM)

4-8 weeks

Shoots

I 3-1 0 weeks BM, 2,4-D (5 FM)

SES (2nd generation)

112 BM, BAP (2. .2 PM), GA3 (1.45 FM) 1 2-5 wee ks

BM, 2,4-D (5 FM) + 6 +4 weeks

SES (3rd generation)

Figure 4.2 Plantlet with hvo stems converted fiom a somatic embryo obtained

fiom green seed, d e r 3 weeks culture on 1.45 pM GA, containing % MS

medium ( 2 . 8 ~ ) .

Figure 4.3 Plantlets converted from somatic embryos d e r 4 weeks culture

on 1/2 BM (top row). % BM containing 1.45 pM GA, (second row). !4 BM

containing 1.45 pM GA, and 2.2 p M BAP (third row), and % BM containing

2.90 pM GA, and 4.4 pM BAP (bottom row; 1 .25~) . Best plantlets are in

the second row.

Figure 4.4 Flowers of greenhouse-grown 3-year-old plants.

A. An inflorescence composed of about 12 flowers ( 1 O . 6 x ) ,

B. A flower. showing 5 petals and 5 anthers (2.8~).

Figure 4.5 An inflorescence developed fiom a somatic embryo-derived plantlet.

A. About 12 flowers in an inflorescence (4.39,

B. Close up of A (17~) .

Figure 4.6 An inflorescence observed in an sornatic embryo-derived plantlet,

having 8 flowers (4.3 x).

Figure 4.7 Flowers of somatic ernbryo-denved plantlets.

A. A flower, having 5 sepals, 5 petals, and 5 anthers (6.4x),

B. A flower, showing 5 petals and 8 anthers (6 .4~) .

Figure 4.8 Anther and pollen grains of somatic embryo-derived plantlets.

A. Pollen grains were released from an anther (1 OOx),

B. Three pollen grains (400~).

Figure 4.9 SEM micrograph of anthers, pollen grains, and surface of pollens of

greenhouse grown 3-year-old plants and somatic embryo (SE) derived plantlets.

Bar=0.38mminAandB,30uminC,38uminDand3uminEandF.

A. C. E. Anther, pollen grain, and surface of pollen of greenhouse grown

3-year-old plant,

B. D. F. Anther. pollen grain, and surface of pollen of SE derived plantlet.

Figure 4.10 Plantlets developed fiom somatic embryos.

A. Plantlets (6 weeks old) grown on !4 BM (2.0x),

B. Plantlet (3.5 month old) with two stems (2.09.

Figure 4.1 1 Three-rnonth-old plantlet developed From a somatic embryo, showing

three well developed shoots, a tap root and lateral roots ( 2 . 6 ~ ) .

Figure 4.12 Roots of greenhouse grown plants (a,) and somatic embryo

denved plantlets (b) after 3 rnonths storage at 3 O C , showing shoot

emergence from the perennating bud ( 2 . 8 ~ ) .

Figure 4.13 Germination of embryos after 9 days culture.

A. A zygotic embryo (ZE) grown on K BM contained L -45 FM GA,,

showing the elongated shoot (2 .8~) .

B. A somatic embryo (SE) grown on 1/2 BM contained 1.45 pM GA3

( M x ) ,

C. A SE grown on !h BM ( 2 . 8~ ) .

D. A ZE grown on !h BM ( 2 . 8 ~ ) .

Chapter 5 Ginsenoside content of regenerated plantlets of American

ginseng

5.1 Introduction

Ginseng root has been used in Oriental medicine since ancient times. Ginsenosides (ginseng

triterpene saponins) are the active constituents of ginseng (Hornok 1992). and six major

ginsenosides can be placed in two classes based on chernical stmcture: the Rb group, which

includes Rb 1, Rb2. Rc. and Rd; and the Rg group. which includes Re and Rgl (Yoshikawa

and F w y a 1987).

In Oriental ginseng, production of ginsenosides in tissue culture using selected ce11

lines has been reported in many studies (Choi et al., 1994; Furuya et al., 1983; Inomata et al..

1993; Odnevall and Bjork, l989a; Yoshikawa and Furuya, 1987: Yoshimatsu et al.. 1996).

Production was affected by various factors including culture rnethods. conditions and age.

and medium composition. In addition, ginsenoside contents of original plants and

regenerated shoots. roots. and plantlets has also been reported (Asaka et al., 1994b; Furuya

et al., 1986).

Using callus and ce11 suspension cultures of American ginseng, Mathur et al. (1994)

showed that the yield and relative distribution of different fractions of ginsenosides were

greatly influenced by culture age. However, there is no published information on the content

of ginsenosides of regenerated materials of Arnerican ginseng.

The objective of this study was to analyse the ginsenoside levels in ernbryogenic

callus and the regenented plantlets which were developed fiom the somatic embryos (SES),

and to compare the contents with roots of field grown plants and in vitro grown seedlings

which germinated fiom zygotic embryos (ZEs).

5.2 Material and methods

1. Plant materiai

Roots offield grown plants

Two and three-yearsld field grown roots of American ginseng were used in this study. The

roots were washed, sliced, and dried at 38 O C , then weighed and ground to a powder using

mortar and pestle. The ratio of fresh to dry weight was about 3 : 1.

Roots of plantlets derivedfiorn somatic ernhyos (SES)

SES induced on auxin containing MS medium were cultured on medium composed of haIf

strength MS salts, B5 vitamins, 1.5 % sucrose. and 3 % gelrite (% BM) and 1.45 pM GA,,

and remained in a growth room at 24 OC under 1 oh-light per day. The roots of 3-month-old

plantlets were dried and ground as that for roots of field grown plants. The ratio of fiesh and

dry weight was about 6: 1.

Ernbryogenic callus

Callus and SES were induced on MS medium supplemented with auxin and cytokinins fiom

cotyledonary explants. The cultures were kept in darkness for five and half months, and

subcultured every 4 weeks. The ratio of fiesh and dry weight was about 30: 1.

Plantlets 1.

SES induced (fiom the ernbryogenic callus descnbed above) on auxin plus cytokinins

contairing medium were cultured on !4 BM supplemented with 1.45 pM GA, and 2.2 pM

BAP under 1 oh-light period at 24 OC. The 2-month-old plantlets were dried. weighed and

ground. The ratio of fiesh and dry weight was about 10: 1.

PIuntlets 2.

SES induced on 2.4-D containing medium were cultured on !4 BM with 1.5 or 3.0 % sucrose

and casein hydrolysate (5 g / L) for 2 weeks; then on !4 BM plus 1.45 pM GA, for 4 weeks;

and then the GA, was removed for another 2 weeks. Plantlets were dried, weighed, and

ground, and the ratio of fiesh and dry weight was about 10: 1.

Seedlings developedfiom ~ygotic embryos @Es)

ZEs were dissected fiom stratified seeds (see Chapter 2 for the details of seed sterilization

and embryo culture), and cultured in petri dish on medium composed of !4 BM for 2 months.

The cultures were kept at 24 OC under l6h-light period a day with a subculturing afier 4

week of culture. The 2-rnonth-old plantlets. about 60 mm high, were dried and ground, and

the ratio of fiesh and dry weight was about 6.5: 1.

2. Ginsenoside extraction

The sarnples obtained from field grown roots and in vitro grown materials were analysed in

Dr. Y. Kakuda's lab (Department of Food Science, University of Guelph). Samples were

extracted using a method developed by Dr. Kakuda (persona1 communication) and similar

to that reported by Smith et al., (1996). The procedure was as follows: 1) approximately 0.5-

125

l g of ground sarnple was extracted with methanol (HPLC grade, Fisher Scientific) on a

Soxhiet apparatus for 4 hrs. The methanol extract was reduced to dryness using a rotary

evaporator, and redissolved in 25 ml water; 2) a 5 ml aliquot of the water solution was ioaded

on a C- 18 Sep-Pak cartridge (Waters Scientific). The Sep-Pak was washed with 10 ml water

followed by 15 ml 30% methanol; 3) the ginsenosides were eluted with 4 ml 100 %

methanol, evaporated to dryness, and the residue dissolved in 2 ml methanol; and 4) each

sample was filtered through a 0.45 nrn nylon filter and was then ready for high-performance

liquid chromatography (HPLC) analysis.

3. HPLC assay

A Waters 600E multisolvent delivery system equipped with a Waters 200 satellite WISP

automatic sarnple injector was used in the analysis. The ginsenosides were separated using

a Beckman Ultrasphere ODS 4.6 mm x 25 cm column coupled with a Beckrnan Ultrasphere

ODS 4.6 mm x 4.5 cm guard column and quantified using a Waters 486 W 1 Vis detector

and Waters baseline 8 15 Chrornatographic Workstation. Ten-microliters of the filtered

samples were injected and eluted From the column at a flow rate of 1.3 ml 1 min using two

solvents (A: Water; B: 100 % acetonitrile) and the following gradient: 0-20 min, 21 %

solvent B: 20-60 min, 2 1 - 42 % of solvent B. After each run, the column was washed with

100 % acetonitrile for 10 min and equilibrated for 15 min with 2 1 % solvent B.

Standards of ginsenosides (Atomergic Chem Metal, 222 Shenvood Ave,

Farmingdale, Long Island, New York, 1 1735) were used to identiQ and quanti& the major

ginsenosides in the plant samples.

Expenment was performed at least nMce for ail the materials except the roots of SE-

denved plantlets because of the smail amount of materiai available.

5.3 Results

Six major ginsenosides. Rgl, Re, Rb 1. Rc, Rb2. and Rd, were detected and quantified in

both roots of field grown plants and in vitro grown materials. The total ginsenoside level of

2- and 3-yeardd field grown roots were 3 1.1 and 41.3 mg / g dry material. respectively (Fig.

5.1 A and B). and two-month-old in vitro grown seedlings had 24.4 mg 1 g (Fig. 5.1 C). Five-

and-half-month old embryogeniç callus had only 1.9 mg / g total ginsenosides (Fig. 5.1 D),

but two months old plantlets which was developed from those SES had 6.6 mg / g total

ginsenosides (Fig. 5.1 E) On the other hand, plantlets that had two different cultural histories

had similar amounts of ginsenosides contents (Fig. 5.1F), while 3 months old roots of

plantlets contained 12.8 mg 1 g of ginsenosides (Fig.S.l G).

Roots of field grown plants contained higher level of Rb group ginsenosides than

those in Rg group while the in vitro cultured materials al1 had higher Rg group ginsenosides

than Rb group, including zygotic embryo (ZE) derived seedlings. The ratio of Rb to Rg

goup was larger than one in field grown roots ( 1.37: 1 and 1.73: 1 ), and smaller than one in

in vitro cultured matenals (0.19: 1 to 0.93: 1 ; Fig. 5.1 H).

Furthemore, in both 2- and 3-year- old roots of field grown plants, Rb1 was the rnost

predorninant ginsenoside, followed by Re, Rc, Rd, Rgl and Rb2 M i l e in vitro cultured

materials were al1 dominated by Re except callus where Rgl was the predominant one.

although the patterns followed were different in seedlings, plantlets and callus, and in

plantlets with different culture histories.

On the other hand. both Re and Rb 1 were the main ginsenosides in roots of both field

grown plants and SE-denved plantlets, and they accounted for > 70 % of the total

ginsenosides. In whole seedlings or plantlets. Re was the only dominant ginsenoside which

accounted for about 50 % of the total amount.

5.4 Discussion

This study showed that the roots of 3-year-old plants contained higher total ginsenosides than

those of 2-year-old plants. This is in agreement with the result of previous studies where the

total content of ginsenosides of American ginseng grown in Ontario increased as the age of

the roots increased from 1 to 4 years old (Court et al., 1996).

Although the contents were lower in SE-derived plantlets, al1 six major ginsenosides

were detected as in roots of field grown plants. In Oriental ginseng, the ginsenoside content

of regenerated plantlets was similar to those of the seedlings, indicating that the regenerated

plantlets and seedlings were similar both with respect to morphology and biosynthesis

(Asaka et al., 1994).

The pattern of ginsenosides was different in plantlets with different culture histones

(both the media for SE induction and for plantlet development were different). although their

total levels were similar (6.5 mg / g). The different patterns also existed between plantlets

I seedlings. roots of field grown plants / roots of SE-derived plantlets, and callus / plantlets.

As mentioned previously. many factors can affect ginsenoside production, such as the

cultural age and condition, and media composition.

The ginsenoside contents of callus , which was cultured under similar conditions to

the regenention of plantlets except that the callus was cultured in darkness, were very low

compared to those of regenerated plantlets. Light / dark condition could affect the production

of ginsenosides. In Oriental ginseng, the stimulating effects of light on ginsenoside formation

in suspension culture (Odnevall and Bjork, 1989a) and callus culture (Choi et al., 1994)

were found, while Furuya et al. (1 983) showed that the effect of light was depended on the

media composition. In addition, Asaka et al. (1 994b) obtained higher ginsenoside amount

in regenerated plantlets than in callus which were grown in similar conditions, and it was

suggested that the ginsenoside production was induced by tissue differentiation rather than

by culture conditions.

In this study, in vitro cultured matends had higher content of Rg group ginsenosides

than Rb group. Similarly, higher levels of Rg group ginsenosides than Rb group were found

in somatic embryos (SES) and the aerial parts of plantlets which were derived from the SES

of Oriental ginseng (Asaka et al., 1994b).

Total ginsenoside content was 3 1 mg / g in roots of 2-year-old field grown plants in

this study, while ZE- derived seedlings contained 24 mg / g total ginsenosides d e r 2 months

of culture on '/z MS basal medium. Furthemore, ginsenoside Re. Rb2, and Rd were higher

in the 2-month-old seedlings than those in the 2-year-old roots (Rb2 was even higher than

that in 3-year-old roots). Higher levels of Rg group ginsenosides was also found in those 2-

month-old seedlings. This could be significant because by culturing ZEs on basal medium

under controlled conditions. we might be able to produce considerable amount of

ginsenosides, which will contain no pesticides, in a relatively short time, "without dirty

hands".

Summary

Six major ginsenosides, Rg 1, Re, Rb 1, Rc, Rb2 and Rd, were detected and quantified by

KPLC in roots of field grown plants and in vitro grown materials of Amencan ginseng. The

total ginsenoside content of 2-year-old roots was 3 1 mg / g dry matter, and the ratio of Rb

to Rg group was 1.37: 1. Rb 1 and Re were the main guisenoside in field grown roots, which

accounted for 70 % of the total amount. The pattern of ginsenosides in the roots was Rb 1>

Re=- R e Rd> Rg 1> Rb2.

In vitro grown materials contained al1 of those six major ginsenosides of field grown

roots, although the total amount were lower (1.9- 24.4 mg / g); the ratio of Rb to Rg group

was smaller (0.19-0.93: I), and Re was the predorninant ginsenoside. The patterns of

ginsenosides in various materials were slightly different. Callus with somatic ernbryos (SES)

on them contained the lowest content in al1 the materials tested, and plantlets which were

developed fiom the SES had 2-fold higher level than that in callus with SE.

Two-month-old in vitro grown seedlings which developed fiom zygotic embryos

contained 24.4 mg / g total ginsenosides. In addition, higher Re, Rb2 and Rd were found in

these seedlings than those in the 2-year-old roots of field grown plants.

Fig. 5.1 Distribution of ginsenoside content and Rb and Rg groups and ratios

in various samples of Amencan ginseng (mean and standard error).

A. Two-year-old field grown roots,

B. Three-year-old field gro wn roots,

C. Two-month-old seedlings,

D. Five- and half-month-old callus,

E. Two-month-old plantlets (plantlets l),

F. Two-month-old plantlets (plantlets 2),

G. Three-month-old roots from somatic embryos,

H. Ratios of Rb group to Rg group in various samples.

GENERAL DISCUSSION AND CONCLUSIONS

American ginseng (Panax quinquefolizim L.) is an economically important crop because of

its highly valued roots (Persons, 1986). On the other hand, ginseng is a slow grower. and

cultivation of ginseng is troublesome (Fisher 1993). Although seeding is the principal

method for propagating ginseng, seeds have immature embryos at &est and 18-22 months

are needed for seed stratification and germination (Proctor and Bailey, 1987).

Somatic embryogenesis, which has been studied in many plant species since it was

fmt reported in carrot (Reinert 1958. Steward et al., 1958), may have potential as an altemate

propagation system for ginseng because of the long stratification penod and high cost of

seeds.

In this study, zygotic embryos were dissected frorn stratified and green seeds.

cultured in vipo and germinated in one weeks of culture. The cotyledons of the seedling were

used as one type of the explants. Tirajoh and Punja (1995) reported that 4-6 months was

required for in vitro germination of stratified seeds of American ginseng at 4 OC. The long

time needed for completion of germination might be due to incubation at low temperature.

the stage of development of the embryo (Proctor and Louttit 1995), and / or an inhibitory

effect of the endosperm.

The important role of exogenous auxin(s) in regulating somatic embryogenesis was

shown in this study. No somatic embryos (SES) were observed on explants which were

cultured on basai MS medium without auxin, however, with 4-6 weeks of exposure to 5 PM

2.4-D, up to 47 % of the cotyledonary explants were able to produce SES. The requirernent

for 2,4-D for iritiation of SES in Amencan ginseng was reported (Li and Guo, 1990), while

SE production occurred on basal medium without growth regulators in Oriental ginseng

(Choi et al., 1996). Furthemore, in the present study, 2.4-D was more effective than

dicamba for induction of SES. inducing more responsive explants and larger number of SES

were obtained. while 15 pM NAA was comparable to 5 FM 2.4-D. Generally, the frequency

and nurnber of SE induction increased as the concentration of NAA increased fiom 5 to 15

pM1 while the best responses with 2,4-D or dicamba were induced with 5 pM awin(s). In

addition. when the combination of 2,4-D and NAA was used in the induction medium, it

was the concentration of 2,4-D that influenced embryogenesis most.

Among the three types of explants tested, shoots obtained fiom SES gave the highest

fiequency and nurnber of SES, the cotyledons next, and the intact zygotic embryos the least

responsive. On the other hand, when dl segments of in vitro grown seedlings were used for

SE induction. 98.5 % of the SES were obtained from cotyledonary explants. The expiant

choice is one of the most important factors determining the success of morphogenesis

(Arnirato 1983), and cotyledons were the most responsive explant to induce SES in peanut

(Murthy et al.. 1995, 1996).

Pretreatrnent, Le., light and GA,, for seedling growth had a strong influence on

subsequent embryogenesis using seedling cotyledonary explant. The addition of GA, into the

medium for seedling growth inhibited embryogenesis. Light did not affect SE induction in

stratified seeds, but stimulated embryogenesis in the case of green seeds.

Combination of auxin and cytokinin(s) have been used for SE production in many

plants including ginseng. In the present study, however, addition of 0.1 - 5 pM of two

cytokinins, kinetin or BAP, to awin (2,4-D, NAA, or dicamba) containing medium did not

affect, or suppressed somatic embryogenesis, although stimulating effect of O. 1 pM BAP was

observed with 10 FM dicarnba supplemented medium in shoot explants afier 8 weeks of

culture. The concentration of cytokinh added to auxin containing medium was crucial (Ahn

et al., 1996; Arya et al., 1993). The types of auxin in the medium, and the expiant type used

are other important factors controlling the response (Takamura et al., 1995; Nadolska-

Orczyk, 1992); however, whereas the two types of cytokinins, kinetin and BAP, did not

exhibit significantly different effects.

Plant regeneration was obtained by germinating SES on haif stren,& MS medium

under light. Inclusion of 1.45 pM GA, in the medium enhanced the conversion rate from 16

% to 80 % after 2 weeks of culture, while addition of 2.2 pM BAP inhibited root formation.

The inhibitory effect of BAP on root formation was shown in previous studies of Arnerican

ginseng (Tirajoh and Punja, 1995) and other plants (Gill et ai., 1995: van Schaik et al., 1996).

In vitro flowering was observed in the plantlets developed from second generation

of SES. (SES induced fiom shoots. which were obtained fiorn first generation of SES). In

Oriental ginseng, in vitro flowering occurred in plantlets derived From first generation of SES

(Chang and Hsing, 1980; Shoyama et al., 1988; Lee et al.. 1 995). Usually, Amencan ginseng

needs a juvenile penod of 2-3 years to reach the flowering age in the field. and in vitro

culture shortened the flowering period significantly.

Field grown roots of Amencan ginseng contain 6 major ginsenosides, Rg 1, Re, Rb1 . Rc, Rb2, and Rd (Court et al., 1996; Li et al., 1996: Smith et al., 1996) . HPLC assay in this

study revealed that regenerated plantlets had the same six ginsenosides, although the total

amount was lower, the pattern was diflerent, and the ratio of Rb group to Rg group was

smaller. Furthemore, callus contained 2-fold smaller arnount of ginsenosides than

regenerated plantlets, indicating that the ginsenoside production was not ody controlled by

culture condition but also by tissue differentiation.

In conclusion, plant regeneration of h e r i c a n ginseng was obtained in 4 months

through somatic embryogenesis. Exogenous auxin(s), their type, concentration and duration,

were critical for SE induction. Over 80 % of the SES converted into plantlets on 1.45 FM

GA, containing medium in 2 weeks. The plantlets contained the sarne 6 major ginsenosides

as those found in the roots of field grown plants.

Future research on somatic embryogenesis in Arnerican ginseng can focus on

fol10 wing two aspects:

1) a more complete range of plant growth regdators can be tested for high SE production and

improved quality of SES;

2) use more green seeds as the explant source, which exhibited similar or even higher

embryogenic potential in this study as that in stratified seeds, to shorten the stratification

time.

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