A Thesis Presented to The Faculty of Graduate Studies Of ... - Cubits

A Thesis
Presented to
The Faculty of Graduate Studies
Of
The University of Guelph -
In partial fulfillment of the requirements
For the degree of
Masters of Science
Oct, 2000
O Kristen Leigh Choffe

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ABSTRACT
MICROPROPAGATION OF ECHI~ACEA PURPUMA L-
Kristen Leigh Choffe Advisor
University of Guelph, 2000 Dr. P.K. Saxena
Echinacea purpureu L. is one of the most cornmon medicinal plants used as an
immunostimulant. The objective of this research was to develop in vitro propagation protocols
for E. purpurea. Both somatic embryogenesis and shoot organogenesis were induced on petiole
sections cultured on a medium containing BAP (benzylaminopurine). Prolific root
organogenesis was observed on etiolated hypocotyl and cotyledon segments exposed to IBA
(indolebutyric acid) and IAA (indoleacetic acid). CaIlus formation was obsexved on hypocotyl
explants exposed to high levels of DA, which when subcultured on BAP supplemented medium
resulted in the indirect differentiation of somatic embryos. Exposure of the etiolated hypocotyls
to TDZ (thidiazuron) in the medium resulted in de novo shoot organogenesis. Together, these
data provide several regeneration systems for the production of consistent plant material for
biochemical analysis and commercial production of Echinacea pttrpurea.

ACKNO WLEDGEMENTS
Dedicatea to the memoqy of qy inother, hi en ne Josce ~ a h o f f e
My sincere appreciation and thanks are foremost directed to Dr. Praveen K. Saxena
without whose faith, trust, guidance and wisdom would not have made this journey a reality. I
would also like to extend my thanks to my advisory cornmittee, Dr. Calvin Chang and Dr. John
T.A. Proctor, whose expenence and knowledge greatly helped in the completion of my thesis.
1 am extremely gratefül to Dr. Susan J. Murch who carefully guided me through and gave
me the strength to survive the pitfalls of my studies. 1 would also like to heartily thank the rnany
people who are an integral part of the Plant Tissue Culture Laboratory for their advise,
assistance, support and bonding sessions throughout my studies. Thank you to Dr. S.
KrishnaRaj, Dr. S heila Chiwocha, Tereza Dan, Mark Garnett, Tannis Slimmon, Skye Campbell,
Mithila Jugulam, Andrew McCartney and Jerrin Victor. My thanks also to Robert Nichols who
was always around to offer his unique sense of humour, patience and advise.
To my sister Kelly Dicks, who has aIways been there to offer her support, comfort and to
share in my joys, and to my father, Donald Choffe, for his faith and love I offer my appreciation
and rny heart. And to my fiancé and soulmate, Edward Galvez, goes my undying devotion for
his patience, love and support as well as for the sacrifices he made for me to achieve this
incredible goal in my life.

TABLE OF CONTENTS
ACKNO WLEDGEMENTS ........................................................................................................................ r'
TABLE OF CONTENTS ......................................................................................................................... r r
LIST OF FIGURES ........ . .............................................................................................................. v
..............................................................................................................................
LIST OF TABLES vzzz
LIST OF A BBRE VIA TIONS .................................................................................................................... i.~
CHAPTER I . LVTRODUCTION ............................................................................................................. 1
Micropropagation Techniques ..................... . .................................................................... - 3
Objectives .................................................................................................................................... 5
Speczjic Objectives ................................................................................. . .................................. 5
CHAPTER 2 . LITERATURE RE V/E w ..................................................................................................... 6
Micropropugution Technology: Poten tial Applications For Production of Quali Echinacea
Prodzicts .................... ,. ......................................................................................................... 6
liztrodziction ................................................................................................................................. 6
Dornzancy and Gernzination ....................................................................................................... 7
Adulîercrtion wirh Misiden trfied Species ..................................................................................... 7
Contamination With Fungi. Bacteria and Insects ....................................................................... 8
L oss of Wild Gemplasnz ........................................................................................................... 10
Need foi- Standardizatiolz .......................................................................................................... I l
In Vitro Propagation ................................................................................................................. 13
In Vitro Cziltzire of Echinacea ................................................................................................... 13
CeZl Szqensioiz Cultures of Echinacea .................................................................................... 14
Agrobacterium Mediated Genetic Transformation ................................................................... 14
. .
11
..
...

Induction of Indirect Regenerarion ........ . ................................................................................ 41
Results ....................................................................................................................................... 42
Direct shoot Organogenesis .................. . .............................................................................. 42
Indirect Somatic Embtyogenesis ............................................................................................... 43
Discussion ................................................................................................................................. 66
CHAPTER 5 . DE Nova ROOT ORGANOGENESIS /N HYPOCOTYL AND COTYLEDON CULTURES OF
ECHINACEA PURPUREA L ................................................................................................................ 68
Abstract ..................................................................................................................................... 68
Introduction ............................................................................................................................... 68
Materials and Methods ............................................................................................................. 69
Esrablishmerir of SteriZe Seedlings ............................................................................................ 69
Explant CzrZture ......................................................................................................................... 70
Results ......................................... . ......................................................................................... 70
Regenemtion of Hypocoryl Explants ........................................................................................ 70
Regeneratioiz of Cotyledon Erplants ........................................................................................ 72
Disctrssion ................................................................................................................................. 89
CHA PTER 6: RESULTS AND FUTURE PROSPECTS .............................................................................. 91
References .............................................................................. . ......................................... 94
APPENDIXI: THE DEVELOPMENT OF STERILIZATlON PROCEDURES FOR ECHINACEA PURPUREA .
ACHENES ....................................................................................................................................... 104
A PPENDIX IL- THESIS PUBLICA TIQNS .............................................................................................. 106

LIST OF FIGURES
CHAPTER I. INTRODUCTION ............................................................................................................ I
Figure 1. Route of regeneration showing indirect differentiation (callus, ce11 suspension and
protoplast) and direct differentiation of new plantlets by organogenesis and somatic
embryogenesis fiom explants. ....................... . ........................................................................ 4
CHA PTER 3. IN VITRO REGENERA TION OF ECHINA cEA PURPUREA L. : DIRECT SOMA TIC EMBR YOGENESIS
AND INDIRECTSHOOT ORGANOGENESISIN PETIOLE CULTURE ............................................................ f 6
Figure 1. Effects of the cytokinin BAP on regeneration of Echinacea pztrpurea petiole
explants. .................................................................................................................................... 2 1
Figure 2. Developrnent of regenerants on Echinacea ptirpurea petiole explants in response to
1 .
BAP (5 prno1.L- ) in the culture medium. ..........................~.................................................... 27
Figure 3. Histological evidence of organogenesis in petiole culture of Echinacea pzirpzirea in
response to BAP ........................................................................................................................ 29
Figure 4. Histological evidence of somatic embryogenesis in petiole cultures of Echirzacea
pzrvpzrrea L. in response to BAP. ............................................................................................ 33
CHAPTER 4. REGENERQT~ON OF ECHMCEA PURPUREA HYPOCOTYL EXPLANTS VIA SHOOT
ORGANOGENESIS AND SOMA TIC EMBR YOGENESIS.. ........................................................................... 38
Figure 1. Effect of the concentration and duration of the cytokinin TDZ on root organogenesis
of Echinacea pztrpurea hypocotyls. .......................................................................................... 46
Figure 2. Induction of shoot organogenesis on Echinacea purpurea hypocotyl explants in
response to different TDZ concentrations and induction period on TDZ-supplemented
medium. .................... . ....................................................................................................... 48

Figure 3. Effects of the auxin NAA and the cytokinin BAP on shoot organogenesis Ecliinacea
pzlrpurea hypocotyl explants after 33 days. ............... . ...................................................... 50
Figure 4. Effects of the auxin NAA and the cytokinin BAP on root organogenesis Echirzacea
ptrrpurea hypocotyl explants der 33 days ..................... . ..................................................... 52
Figure 5. Callus formation on an Echinaceapurpurea hypocotyl on 100 pmol-~-l BA after 4
weeks of culture- ............................ . .................................................................................. 54
Figure 6. Effects of the auxin IBA and the cytokinin BAP on formation of root organogenesis
from callused Echinacea purpurea hypocotyi explants after 33 days. ..................................... 56
Figure 7. Effects of the auxin B A and the cytokinin BAP on formation of sornatic
embryogenesis from callused Echinacea purpurea hypocotyl explants after 38 days. ............ 58
Figure 8. Different stages of somatic embryogenesis formed fiom callus induced by culture of
Echinacea hypocotyl explants on 100 pmol-L-1 IBA and subcukured on 7.5 pmol-L-1 BAP
after 3 8 days of subculture with BAP. ...................................................................................... 60
Figure 9. Globular stage somatic embryos formed fiorn hypocotyl explants of Eclziiîacea
pzirpurea L. cultured on 100 pmol-L" IBA and 5 pmol-~-l BAP after 2 1 days of subculrure
with BAP ...................... . .................................................................................................... 62
Figure 10. Heart shaped somatic embryos formed corn hypocotyl explants of Echinacea
purpureu L. cultured on 50 pmol-~'l IBA and 2.5 pmof-~-' BAP afler 21 days of subculture
with BAP. .................................................................................................................................. 64
CHAPTER 5. DE NO VO ROOT ORGANOGENESIS IN HYPOCOTYL AND COTYLEDON CULTURES OF
EC~ACEA PURPUREA L. ............................................................................................................... 68
Fiawe 1. Root organogenesis in cultured hypocotyl explants of Echinacea purpzu-ea L. after
28 days in culture. ..................................................................................................................... 73
vi

Figure 2. Root organogenesis of Echinacea pu~urea hypocotyl explants cultured on NAA
after 28 days in culture ............................................................................................ 75
Figure 3. Root organogenesis of Echinaceapztrpurea hypocotyl explants cultured on BA.. 77
Figure 4. Root organogenesis of Echinacea puvurea hypocotyl explants cultured on M.. 79
Figure 5. Effect of IPA concentration on root origin (direct or indirect regeneiation) in
Echinacea purpurea hypocotyl explants der 28 days in culture ............................................ 8 1
Figure 6. Root organogenesis in cultured cotyledon explants of Echinaceaprtrpitrea L. afier
28 days in culture. .........,... ................. - ......................... . .......................... ............... .. 83
Figure 7. Effects of the auxin IBA on root organogenesis of Echinacea piirprtrea cotyledon
explants after 28 days of culture. ......................................................................... ........... . 85
Figure 8. Effects of the auxin IAA on root organogenesis of Echinacea purpzrrea cotyledon
explants after 28 days of culture. .................................... . ............................................... 87
vii

LIST OF TABLES
CHAPTER 2. LITERA TURE RE VIEW .................................................................................................... 6
Table 1: Chernicals produced in achenes and seedlings of Echinacea purpurea. E.
angust~yolia and E. pallida ......................................................................................................... 9
Table 2: Various substances found in Eclzinac~a species and their proposed pharmaceutical
properties ............... . ......................................................................................................... t 2
CHAPTER 3. IN VITRO REGENERA TION OF ECHINACEA PURPUREA L. : DIRECT SOMA TIC ElMBR YOGENESIS
AND INDIRECT SHOOT ORGANOGENESIS IN PETIULE CULTURE. .............. ,. ........................S......-......... 16
Table 1. Effects of the auxin NAA and the cytokinin BAP on formation of somatic
embryogenesis, shoot and root organogenesis of Echinacea purptiren petiole explants. ........ 23
Table 2. Effects of the auxin 2,4-D and the cytokinin BAP on formation of sornatic
embryogenesis and root organogenesis of Echinacea purpurea petiole explants. ................... 24
Table 3. Effects of IAA and TDZ on regeneration of Echinaceapurpzirea petiole explants. .. 23
Table 4. Effects of TDZ concentration and duration on formation of somatic embryogenesis,
shoot and root organogenesis of Echinacea purpurea petiole explants. .................................. 26
-~PPEND/XI: THE DE VELOPMENT OF STER(LIZ4 TION PROCEDURES FOR ECHINACEA PURPUREA L.
ACHENU. .................................................................................................................................... 104
Table 1 : Sterilization methods and results for Echinacea purpurea achenes. ....................... 105

LIST OF ABBREUA TIONS
-y 7 4-D 2,4-dichlorophenoxyacetic acid
BAP benzylaminopurine
FAA fonnalin/glacial acetic acid
GA3 gibberellic acid
M indoleacetic acid
IBA indolebutyric acid
MS Murashige and S koog Medium ( 1963)
PGR P Iant Growth Regulator
PPM Plant Preservation Mixture Plant Ce11 Technology, inc. Washington, D.C.
NAA naphthaleneacetic acid
SAS Statistical Analysis System
TDZ thidiazuron m-phenyl-NY(1.2,3-thidiazol-5-yl)urea]

CHAPTER 1. I~ODUCTION
The past 20 years have seen a rapid nse in phytopharmaceutical consumption.
According to the Canadian Phannaceutical Association, the Canadian herbal medicine
market was valued at $1 50 million in 1995 and is growing at an annual rate of 15%. A
1993 survey accounted for 20% of Canadians who use some form of alternative
medicine. As well, according to the New York Times and the Arnencan Pharrnaceutical
Association, greater than one third of Americans are using herbs for health purposes and
1999 saw sales in this area approaching $4 billion (Barrett et al., 1999). Many
estabIished pharmaceutical companies are now looking into manufacturing
phytomedicinal and nutraceuticals products. However, there is a need for some kind of
quality control to supply the market with standardized and consistent plant material.
Echinacea purpzirea L. is one herbal product that has considerabIe consumer
appeal as cold and flu prevention supplements. Echinacea products are ranked arnung
the top 10 herbal products (Clarke, 1999) and represent 9.9% of the market share of the
medicinal herbal industry (Li, 1998). Although Echinacea products are widely sold in
North Arnerica no standardization exists for the plant material in terms of "suspected"
medicinal compounds produced ftom secondary metabolites. Large inconsistencies can
occur in crop content or composition due to year-to-year variability, insect or disease
infestation or other environmental factors. Moreover, the effects of these influences are
manifested over the 3 - 4 year production time of a harvestable product (Li, 1998).
As with many herbal preparations, the characterization of active ingredients and
commercial production of Echinacea species has been limited by a range of issues
including: a) contamination of the plant material with insects, h gi and bacteria, b) the

length of time required for production of a saleable product, c) plant-to-plant and year-to-
year variability in the active components of the plant material, and d) the lack of pure
standardized plant material for biochemical analysis. In addition, the medicinal content
of phytopharmaceutical plant preparations often varies due to adulteration OF medicinal
preparations with misidentified plant species, consumer fkaud, a lack of adequate rnethods
for production and standardization of the crop, and a lack of understanding of the unique
plant physiology or efficacy with human consumption.
Micropropagation Techniques
Plants have the unique capability to develop into complete plants fiorn a single
cell. This phenomenon is termed totipotency and each plantlet developed £tom these
cells is likely to be similar to the parent plant (Thorpe, 1994). Micropropagation of
phytomedicinal species exploits this ability and, by careful manipulation of plant growth
hormones and nutrients, provides us with the ability to produce rnany identical or clona1
offspring expressing the sarne or greater concentrations of their valuable metabolites.
In the micropropagation process, srnall pieces of tissue called explants are excised
from seedlings or plantlets and exposed to an induction medium that satisfies al1 of the
requirements for plant growth and development. Disrupting the connection of plant cells
with the matemal tissue allows for the manipulation of the developmental pathways
(Steward, 1961). Addition of phytohormones to the culture media redirects the growth
and differentiation of somatic cells (Skoog & Miller, 1957). New ce11 proliferation and
differentiation in criltured plant cells can occur in two different developmental pathways:
a) organogenesis or b) somatic embryogenesis. The organogenic mode of development
results in renewed generation of shoots and/or roots fiom cultured tissue to asexually

produce organs and eventually whole plants. Somatic embryogenesis is also an asexual
method of propagation and the resulting clones are genetically identical unlike sexually
produced embryos, which are recombination products of individual male and female
garnetes. In both organogenesis and sornatic embryogenesis, the differentiation is either
direct or indirect. Indirect morphogenesis is defined as the formation of callus on
explants and the subsequent development of shoots, roots or somatic embryos (Sharpe et
al., 1980). Development of callus or ce11 culture is the result of a dedifferentiation or
reversion of the plant cells to the meristematic phase of ce11 development. In contrast,
direct differentiation is the development of organogenesis or somatic embryogenesis
directly £tom the explant tissue without a callus (dedifferentiation) stage (Fig. i).
The developmental route and fiequency of regeneration in any tissue culture
system is dependent on several factors: a) selection of an appropnate explant, b)
preparation of the explant, c) supplementation of plant tissue culture media with the
optimal combination of growth regulating compounds and amendments, and d)
optimization of environmental conditions for the development of regenerants (Thorpe
1994; Skoog & Miller 1957).

Plantlet
Figure 1. Route of regeneration showing indirect differentiation (cailus, cell suspension
and protoplast) and direct diEerentiation of new plantlets by organogenesis and somatic
embryogenesis tiom explants.

Objectives
The main objective of this research was to develop and optimize efficient
micropropagation systerns for the phytomedicinal species Echinacea pziipzirea L. These
systems will provide a means to obtain large amounts of sterile, consistent plant material,
superior in their Ievels of active ingredients over a reduced time frame when compared to
conventional practices.
Speczjk Objectives
To accomplish the main objective the following approaches were taken:
To evaluate the morphogenic potentiaI of various Echinacea purpuvea explants
To use auxins and cytokinins to develop prolific root and shoot cultures using
both hypocotyi and petiole explant tissues of Echinacea pzrrpztrea
To develop and maintain callus cultures for Echinacea pzrrpurea and to examine
the ernbryogenic potential of the callus

Micropropagation Technology : Poten tial Applications For Prodziction of Qrralip
Echinacea Products
Introduction
Echinacea products are among the best-selling herbal remedies in North America.
Although Echinacea has been used by native Americans since the lsth century, the
popularity of Echinacea throughout the world can be found originating back to early
190OYs, when Dr. H.C.F. Meyer, a Nebraska patent medicine purveyor and creator of the
phrase "snake-oil salesman", offered to let himself be bitten by a rattlesnake in order to
prove the curative ability of Echinacea (SrnaIl & Catling, 1999; Kindscher, 1989). In
1998 Echinacea was rated highest among the top ten medicinal herb products (Klausner.
1998) and sales in 1996 were $1.2 million, an increase of 53.4% fkorn the previous year
(Richman & Witkowski, 1997). Echinacea products have also been recognized by the
health protection branch of Health Canada for its effectiveness in easing sore throats
(Driedger, 2 997).
The introduction of powerfûl and easily obtained antibiotics in the 1930's and
40's caused a rapid decline in the popularity of Echinacea products but the products
began to regain their appeal in the 1 980's and 90's (Kabaganian et al., 1 999). S ince there
is no known cure for the cornmon cold, traditional rnedicines are being used to help
alleviate cold syrnptoms (Driedger, 1997). Echinacea has a long history of efficacy in
immune system stimulation and vimially no toxicity (Mengs et al., 1991). Ecliinacea
products are available in a wide variety of forms including liquid capsules, chewable
tablets, tea bags, tinctures and powders. The suggested dosage to reduce the severity of

colds is lg/day of rhizome and root products and 8 - 9 mVday of juice produced from
aerial plant portions. Cycles of 10 days on and 4 days off (Whatley, 1999) for no longer
than 6 to 8 weeks (Ness et al., 1999) have also been suggested for full effectiveness.
At the present time, Echinacea products are denved ffom conwentional fieId-
grown plants. In spite of the commercial success of these products, there are many
challenges associated with the growth and production of Echinacea. For instance, as
with many other field-grown medicinal plants, uncontrollable environmental effects such
as rainfall or drought can lead to plant-to-plant or year-to-year variatimn in yield and
amount of secondary metabolites produced by the plant species in question (Murch et al.,
2000).
Donnancy and Germination
Echinacea anguîtifolia has been shown to have very low germination levels and
variations in emergence times due prirnarily to seed dormancy (Small & Catling, 1999;
Baskin et al., 1992). The priming and stratification of the seeds can partiailly compensate
for this problem. Practices such as priming in a potassium phosphate buffer or with
giberellic acid have shown an increase in seed germination by as much as 266% (Pill &
Haynes, 1 996).
Adzilteration with Misidentifed Species
The nine species of Echinacea are often confirsed due to discrete differences in
morphology and chernistry, which are not always apparent to an untrained eye. An
example is seen in extensive literature on E. angustifolia in which it was consistently
referred to as E. pallida (Baskin et al., 1994). In several instances Ecltinacea purpurea
products have been contaminated with Parthenizim integrifoo[iitim (compositae; Bauer,

1998; Hobbs, 1989) as well as contamination with other Echinacea species (07Hara et
al.; 1998; Hobbs, 1989). Although these species have been proven to be non-toxic, the
medicinal effects of the products may still be compromised. For example, commercial
preparations of E. angustifolia and E. purpzïrea have been found to contain Echiriacea
pallida as a result of misidentification due to similar plant morphology (Small & Catling,
1999; Hobbs, 1989). The profile of active constituents in E. pallida is different than that
of either E. angustifolia or E. purpuren and this species is less comrnercially valuable
(Hobbs, 1989). The most common way of differentiating Echirtacea species is the colour
of the pollen. The flowers of E. purpurea and E. nngustifolia produce yellow pollen
while E. pallida flowers typically have white pollen (SrnaIl & Catling, 1999). The
misidentification of Echinacea species can have senous consequences since the species
contain different chernical profiles (Schulthess et al., 1991). Biochemicals. which
separates the three most notable medicinal species of Echinacea species, are summanzed
in Table 1.
Coniam inaiion With Fungi, Bacleria and Insects
Since Echinacea is commonly produced in field conditions, commercial
producers have experienced loss as a result of fungi, bacteria andor insect infestations.
Another serious problem associated with bacterial and fimgal contamination can be the
destruction or alteration of medicinal compounds by the microorganisrns (Bemath, 1986).
Additionally, the bacteria, Fungi and insects associated with field-produced Echinacea
can contaminate commercial products and as a result, consumers may be exposed to
health risks.

Table 1: Chernicals produced in achenes and seedlings of Echinacea purpurea, E.
angrtstifolia and E. pallida (Schulthess et al., 1991).
Species
E. purpurea
Tissue
Achene
Achene
Achene
Root
Compounds specific to each species are denoted with *
Chernical Compounds
B-farnescene*
unidentified compound X*
dodeca-2Ey4E,8Z, 1 OE(1 OZ)-tetraenoic acid isobutylamide
1,8-pentadecadiene*
derivative of gerrnacrene D*
dodeca-2E,4E,8Z, 1 OE(1 OZ)-tetraenoic acid isobutylamide -
(much lower quantities)
Carvcementhene*
caryphyllene*
gerrnacrene D*
trideca-2E,7Z-diene- 10,12-diynoic acid isobutylamide*
dodeca-2E,4Ey8Zy 1 OE(1 OZ)-tetraenoic acid isobutylamide
trideca-2E,7Z-diene-10,12-diynoic acid isobutylamide*
dodeca-2E,4Ey 10E-trien-8-ynoic acid isobutvlamide*

Weed Compet ition and Adtzlterarion
Weed interference may not only inhibit the establishment and growth of
Echinacea fields but may also be a source of contamination when plant materia1 is
harvested conventionally. The use of specific herbicides on weed species found mon;
Echinacea stands has been shown to be effective for weed control. Beran et al. (1999)
found that the use of imidazolinone herbicides effectively controlled invading weed
species without hanning the growth of Echinacea plants. A Metolachlor mixture with
sirnazine, isoxaben and oxadiazon was also shown to be effective for control of weeds not
only on natural stands but also in nursery production of Echinacea (Derr, 1993).
However, since Echinacea is commonly sold in health food stores as a "natural" product,
growers are looking for alternate methods for organic production and consurners may not
accept the use of herbicides for this crop.
Loss of Wild Germplasrn
The increased popularity of Echinacea products has also brought about an
increase in small markets selling herbai products. Many of these products contain plant
material coUected fiorn the wild, and these plant collectors rnay not discriminate between
species of Echinacea or the amount of matenal being harvested. This practice has
become a threat in the persistence of many species of Echinacea, some of which such as
E. tennesseensis have already been placed on the endangered species list. To meet the
dernand for Echtnacea products and alleviate the harmful effects of overharvesting,
increased and more efficient cultivation and propagation methods are necessary
(Kindscher, 1989).

Need for Standardriation
Currently, there is very IittIe scientific information and therefore limited
standardization available for medicina1 plants in general and Echinacea speci fically . As
well, the information that is available cm be complex and comtradictory. As a result,
major discrepancies occur in levels and ingredients present in herbal products. Many
companies standardize their products to the amounts of echinacoside present which is a
caffeic acid denvative found mainly in roots of E. angustfoZia and E. pallida but does not
possess irnrnunostimulatory effects and only low antibactenal and antiviral activity
(Bauer, 1998). Pinpointing one specific ingredient has proven to be extremeiy difficult.
For instmce as many as 15 alkamides, which are presumed to b e responsible for the anti-
inflammatory action of the species, have been identified in the roots of Echinacea
angustifoZia and 11 alkamides in the roots of E. prclpurea @amer, 1998). Cichoric acid
found in roots of E. pztrpurea as well as numerous polysaccharides isolated fiorn
Echinacea species have been found to possess immunostirnulatory properties (Bauer,
1998). In many instances, it is the relative balance of several comstituents that determines
the effkacy of Echinacea products. The most highly characterized chemical constituents
of Echinacea species are summarized in Table 2. Optirnizatiom of specific medicinally
active biochemical profiles of Echinacea is desirable but may require extensive research
as the efficacy of the individual components is not fûlly established as yet (Wagner,
1999).

Table 2: Various substances found in Eclzinacea species and their proposed
pharmaceutical properties.
Species
E. purpztrea
E. purpztrea
E. purpztrea
Echinacea spp.
Echinacea spp.
Substance '
Alkamides
Root extract 1 Imrnunos tirnulatory
action
Arabinogalactan Macrophage
activation
Arabinogalactan Immunornodulating
activity
Polysacchaxides Protection against
systemic infections
Polysaccharides Phagocyte system
activation
1
Properties
Anti-inflammatory
Polysaccharides Anti-inflammatory
I
Unpurified pressed 1 Imunostiniulatory
juice
action
Arabinose and
galactose
Immunomodulating -
activity
Ec hinacoside, Preventionheatment
chicoric acid,
cynarine, caffeic
acid and
chlorogenic acid
of skin photodarnage
Reference
Muller-Jakic et al.,
1993
Tragni et al., 1988
&1986
Rehman et al., 1999
Leuttig et al., 1989.
Stimpel et al., 1984
Schollhorn et al.,
1993
Roesler et al., 199 1
Roesler et al., 199 1,
Steinmuller et al.,
1993
Burger et al., 1997
Willigmann et al.,
1993
Facino et al., 1995 1

Irz Vitro Propagation
One solution to the problems faced by the phytopharmaceutical industry is the
development of in virro systems for the production of medicinal plants. In vitro
production of plants has several advantages: a) plants are grown in sterile, standardized
conditions therefore eliminating adulteration with fungi, bactena, insects, or any other
plant species; b) wild gennplasm may be replenished and conserved, c) dormancy periods
are reduced or eliminated; d) individual superior plants cm be identified and clonally
propagated; e) plant material is consistent and therefore, precise biochemical
characterizations can be achieved; and f) eventually protocols can be developed for the
improvement of the crop through genetic manipulation.
Individual plant cells possess the unique capacity to regenerate and ultimately
produce whole plants in tissue culture without undergoing the process of sexual
recombination, a phenornenon known as totipotency (Thorpe, 1994). The development
of tissue culture techniques relies on this regeneration ability by shifting the growth
direction of the cells in various plant organs in ways such as the inclusion of plant growth
regulators in the induction media and manipulation of the culture environment (Thorpe,
1994). Optirnization of the various media components will ultimately result in the
production of masses of uniform clones of superior individual plantlets.
In Vitro CuZtttre of Echinacea
There is currently only Iimited information available with respect to the in vitro culture of
Echinacea. The research approaches that have been attempted include the establishment
of cell suspension cultures and hairy-root cultures.

Cell Suspension Cultures of Ech inacea
Many of the in vitro techniques developed for Eclzinacea species have centered
around the development of ce11 suspension culhires. In general cell cultures allow for the
continuous harvest of plant chemicals and have been effective for the extraction of many
valuable products (Alfemann & Petersen, 1995). Examples of cell culture derived
medicines are Taxol, an anti-cancer drug fiom Tarus sp. (Jaziri et al., 1996), L-DOPA,
used for the treatment of Parkinsons disease, £tom Mucuna pruriens and the sedativelpain
killer codeine fiom Papaver sornn$erurn (Pras et al., 1995). Echinacea ce11 cultures were
produced by fust culturing stem explants into a Iiquid medium supplemented with NAA
and kinetin (Rether et al., 1990) followed by biweekly subcultures on the same medium.
Cells cultured in this way generate a continuous supply of Echinacea ceils, which
produce the same biochemical compounds as were found in intact plant organs (Rether et
al., 1990). These cultures were then used for the extraction and analysis of their active
biochemical constituents and consequent testing for mammalian efficiency (Steinmuller
et al., 1993; Leuttig et al., 1989; Tragni et al., 1988; Wagner et al., l988). The
development of efficient cell suspension cultures has lead to the development of a
15,000-L fermenter allowing for the production of Echinacea polysaccharides on an
industrial scale for use as purified drug products (Bauer, 1998).
Agva bacterium Mediated Genetic Transformation
Agrobacterium rhizogenes transformation has been used for the development of
hairy root cultures of Echinacea (Trypsteen et al., 1991). A. rhizogenes inserts a
fragment of DNA into the host genome which results in the rapid growth of a prolific root
system (Nilsson & Olsson, 1997). This system is a widely acceptable technique for the

transformation of many valuable medicinal root crops to promote a profuse root system
providing a more profitable harvest of the secondary metabolites of interest. The
establishment of an efficient system for hairy root cultures of Echinacea allowed for the
growth of an increased volume of roots and the mass production of the various active
polysaccharides in sterile cultures (Trypsteen et al., 1991).
In spite of these research efforts the establishment of ce11 suspension and hairy-
root cultures has not significantly impacted the commercial production of Echinacea. It
is possible that intact tissues or plantlets are required for the optimal production of
medicinal components. As well, clona1 production of large nurnbers of superior
individual Echinncea genotypes has the potential to provide growers with an improve3
gempiasm. Therefore the primary objective of this study was to develop in virro
regeneration protocols for Echinacea. The in vitro grown plant material is ideal for the
identification and standardization of medicinally active compounds and therefore
represents the first step in the production of consistent, high-quality Echinacea products.
Further research aimed at selection of superior individual plants with preferred
biochemical profiles will lead to the ability to clonally propagate the species, increase the
levels of active constituents and improve the overall consistency and efficacy of the
Echinacea products. Thus the development of micropropagation techniques and
implementation on a commercial scale not only has the potential to alleviate many of the
problems associated with the conventional practices of field production but wiII also
enable consumers to be able to buy standardized preparations of Echinacea without
concern for the quality of the product.

CHAPTER 3. IN VITRO REGENERATION OF ECHINACEA PURPUREA L+: DIRECT SOMATiï
EMBR YOGENESLS AND INDIRECT SHOOT ORGANOGENESIS IN PETIOLE CULTURE
A bstract
An in vitro propagation system was developed for Echinacea purpzwea L. (purple
coneflower), a medicinal plant cornmonly used in the treatrnent of colds, flu and related
ailments. Echhacea seed were found to be contarninated with systemic fimgi and
therefore an optimized minimal concentration of Plant Preservation Mixture (PPM) was
incorporated in the seed germination medium to recover stenle seedlings. Regeneration
was induced on petiole explants £tom two-month-old stenle seedlings cultured on
medium supplemented with benzylarninopurine (BAP) or thidiazuron (TDZ) in
combination with indoleacetic acid (IAA). Two distinct forms of regeneration were
identified in cultured petiole explants with histological and morphological observations
viz. the direct formation of somatic embryos on the epidermis and the de novo
developrnent of shoots from callus tissues formed in subepidermal ce11 layers.
Introduction
Echinacea products are cuxently among the best-selling herbal remedies in North
Amenca and have been for several years (Schardt, 1998). Preparations of Echinacea sp.
have historically been used for the treatment of cornmon human ailments such as colds
(Kindscher, 1992). Extracts and dned samples are taken from the mot of Echinacea
species, a crop that requires about 3 years to produce a saleable product (Li, 1998).
Recent technological advances have allowed researchers to analyze some of the
medicinally active compounds present in Echinacea sp. and to speculate on their modes
of action. Complex polysaccharides, such as arabinogalactane and xyloglucan, extracted

from the roots of different Echinacea sp. have been found to stimulate rnamrnalian
immune systems (Coeugniet and Elek, 1987) and to act as anti-inflammatory agents
(Tragni et al., 1988). These phytochemicals have been found to activate mammalian
macrophages (Stimpel et al., 1984), phagocytes (Steinmuller et al., 1993) and to stimulate
the production of lymphokines (Coeugniet and Elek, 1987). In addition, several other
potentially active ingredients of Echinacea including caffeoyl derivatives such as
echinacoside, chlorogenic acid, chicoric acid, cynax-in, and caffeic acid, which is known
to prevent skin photodamage (Facino et al., 1995).
As with many herbal preparations, characterization of active ingredients and
commercial production of Echinacea have been limited by a range of issues including: a)
contamination of the plant material with insects, h gi and bactena, b) the long period of
tirne required for production of a saleable product, c) plant-to-plant and year-to-year
variability in the active components of the plant material and d) the lack of pure
standardized plant material for biochemical analysis. The establishment of a system for
in vitro culture of Echinacea will help to address many of these issues. Therefore, the
principal objective of the current research initiative was to develop an efficient
micropropagation system for Echinacea purpurea L..
Methods
Esta bZish rn en t of Sterile SeedZings
Echinacea purpurea L. achenes were sterilized by irnmersing in 70% ethanol for
30 sec, soaking in a 5.4% sodium hypochloride solution containing one drop of Tween 20
per 500 ml for 18 min, followed by three rinses in sterile deionized water. Fungal
contamination of the seeds warranted the supplementation of the germination medium

with Plant Preservation Mixture (PPM, 1998). To determine the lowest concentration of
PPM which would be biostatic to fungal growth, various concentrations (1, 2, 3, 4 and 5
ml-L") were included in the water agar (8 g-~-') medium. Stede seeds were geminated
in a growth cabinet in 24-hour darkness at 24°C. After 14 days, four stenle seedlings
were subcultured per Magenta box in a medium containing MS salts (Murashije and
Skoog, 1962), B5 vitamins (Gamborg et al., l968), 30 g-~-' sucrose and 3 g.~-' gelrite.
Petide Culture
Mer 2 months in culture, petiole explants, 2 cm in length, were excised from the
stenle Echinacea purpurea L. seedlings and cultured onto the same medium
supplemented with various combinations of plant growth regulators (PGRs). A series of
expenments was conducted to compare the regeneration inducing potential of
indokacetic acid (IAA: 5 and 10 pmot.~'l), thidiazuron (TDZ: 0.5, 1, 5 and 10 pmol-L-' ),
naphthaleneacetic acid (NU: 5 and 10 ~~oI-L-'), berizylaminopunne (BU: 1, 2.5, 5,
7.5, 10, 12.5 and 15 pmol-~-') or 2,4-dichlorophenylacetic acid (2,4-D: 5 and
10 pmol-L-'). IAA and TDZ were also added to the media together. Treatments were
incubated in a growth cabinet with a 16 hour photoperiod under cool white light (40-60
pmol pmc2-s-'). Regeneration was quantified after 25 and 33 days for al1 petiole cultures.
After 33 days, the regenerants were excised fiom petioles and subcultured ont0 basal
medium in test tubes for the development of plantlets.
Light Microscopy
Petiole explants cultured on medium supplernented with 5 pmol-~*' BAP were
harvested at 0, 3, 5, 7, 14, 21, 28 and 35 days of culture. Samples were immediately
fixed in formalin/glacial acetic acid (FAA) and 50% ethanol mixture (5:5:90: v/v/v).

Proper and rapid fixation of the sample was ensured by vacuum infiltration of the
samples at -20 kPa for IO min. The samples were then dehydrated through a graded
tertiary butanol series and embedded in paraffin wax. Transverse 8 pm thin sections
were cut using an ultra microtome (Porter-Blum uItra microtome MT- 1, Ivan Sorvall Inc.,
Connecticut, USA) and stained with alcian green and safianine (Jensen, 1962). The
sections were observed under a compound light microscope (Zeiss, Germany).
Data AnaZysis
For al1 experiments, the treatrnent consisted of 10 plates per treatment with 6
explants per plate. Statistical analysis was carried out using the Student Newman-Kuells
(SNK) means separation test based pm a GLM mode1 of SAS (Statistical AnaIysis
System Inc., 1995). The means separation test choosen was SNK as previously
recornrnended for in vitra protocols (Mize et al., 1999). This is a protected mode1 as the
F value was close to one in al1 treatments and therefore the mean did not differ
substantially (Snedecor & Cochran, 1989).
ReszcZts
EZirnination of Sysremic Fungal Contamination
Echinacea seeds had systemic fungal pathogens that were not completely
destroyed by the seed sterilization protocol using sodium hypochlorite. Addition of PPM
to the seed germination medium effectively circumvented the problern of contamination.
The optimal concentration of PPM was determined to be 3 ml-L-'. At higher
concentrations, the Echinacea seedlings were significantly shorter and their hypocotyls
appeared sninted while concentrations lower than 3 rnl-~-' did not effectively elirninate
fùngal growth.

Regeneration F m Petiole Eqlants
Regeneration (shoots andlor somatic embryos) was induced on petiole explants on
media containing BAP or the combination of TDZ and IAA. Media containing 1 - 15
pmol-~-l BAP as the sole PGR was found to induce de novo shoot formation at al1
concentrations (Fig. 1) and 80 - 90% of the explants responded to the treatment regardless
to the BAP concentration. The optimum level of BAP was 2.5 pnol-L-', which yielded
an average of 8.1 regenerants per explant in 33 days. Application of BAP in cornbination
with NAA or 2,4-D inhibited regeneration (Tables 1 and 2). When 2,4-D was used alone
regeneration was inhibited (Table 2). Petiole explants induced with NAA alone at 5 or 10
pnol-L-' formed an average of 2.2 roots per explant and only 54.5% of the explants
responded (Table 1). Similarly, about 90% of the explants showed root formation with
IAA (5 and 10 Itmol-~-l) alone whereas regeneration was achieved in >95% of the
cultures when IAA was combined with TDZ or when the medium was supplemented with
TDZ alone (Tables 3 and 4).

Figure 1. Effects of the cytokinin B AP on regeneration of Echinacea purpurea petiole
explants.
Statistical differences were assessed by Student Newman-Kuells mean
separation test after 33 days of culture.

O
Con
5 7.5 10 12.5
Benzylaminopurine (pmc

Table 1. Effects of the auxin NAA and the cytokinin BAP on formation of somatic
embryogenesis, shoot and root organogenesis of Echinacea purpurea petiole expiants.
Statistical differences assessed by the Student Newman-Kuells mean separations
test afier 33 days of culture.
NAA BAP Number of Number of Number of
Concentration Concentration ernbryosl shoots/ roots/
(09 (PM) petiole petiole petiole
abc Values within a column with different superscripts are significantly different
(P

Table 2. Effects of the auxin 2,4-D and the cytokinin BAP on formation of somatic
embryogenesis and root organogenesis of Echinacea puqpurea petiole expiants.
Statistical differences assessed by the Student Newman-Kuells mean separations
test after 33 days of culture.
NAA 2,4-D Number of Number of
Concentration Concentration embryod roots/
(PM) (PM) petiole petiole
abc Values within a column with different superscripts are significantly different
(P

Table 3. Effects of MA and TDZ on regeneration of Echinacea purpruea petiole explants.
Statistical differences assessed by the Student Newman-Kuells mean separation test after
33 days of culture.
M TDZ Number of Nurnber of
Concentration Concentration regenerantsl rootslpetiole
(CLMI (PM) petiole
O O 0.0" 0.0"
ab Values within a column with different superscripts are significantly different
(P

Table 4. Effects of TDZ concentration and duration on formation of somatic
embryogenesis, shoot and root organogenesis of Echinacea purpurea petiole explants.
Statistical differences assessed by the Student Newman-Kuells mean separations test after
3 3 days of culture.
TDZ
Concentration
(PM)
Duration Numberof Numberof Numberof
on TDZ shoots/ embryod roots/
(day s) petiole petiole petiole
a Values within a column with different superscnpts are significantly different
(Pc0.05).

Figure 2. Development of regenerants on Echinacea purpurea petiole explants in response
to B AP (5 pmol-~-') in the culture medium.
A. Callus formation was clearly visible on some petioles and shoot regenerants appeared at
the callus interface. B. Individual shoot regenerant on the explant with weIl defined leaf
initials afier 33 days of culture. C. Late cotyledonary stage somatic embryo on a petiole
explant after 33 days of culture. D. Complete plantlets developed £tom somatic embryos
grown on basal medium after 2 rnonths.

Figure 3. Histological evidence of organogenesis in petiole culture of Echinacea ptzrprwea
in response to BAP
A. Pencha1 ceIl division in the subepidermal layers of the petiole explant at day 3. Bar
= 20 Fm. B. Formation of promeristematic centers in the callus tissue by day 14
(arrowheads). Bar = 680 Pm. C. A closer view of the promeristernatic centers observed
on the callus tissue at day 14 revealed cells which were smaller in size and had a dense
cytoplasm with prominent nuclei. Bar = 50 Fm. D. These promeristematic centers
developed were further developed into dome shaped meristem zones which were well
defined by day 21. Bar = 50 p m E. Development of a shoot meristem and the leaf
primordia by day 21 (arrowhead). Bar = 320 Fm. F. A well developed shoot bud
surrounded by leaf primordia at day 28. Note: Trichornes were observed to be associated
with the leaf primordia and xylem elements were present at the base of the shoot bud. Bar
= 166 Fm.

Visual and histological observations of the cultures revealed two distinct routes of
morphogenesis resulting in the formation of both somatic embryos and shoots in response
to BAP (Figs. 2, 3 and 4). Figure 2 illustrates the various stages of plant regeneration
observed in Echinacea purpurea L. petiole cultures. Some petiole explants exposed to
BAP dedifferentiated to fonn callus fiom which de novo shoots arose (Fig. 2A and B)-
Other petiole explants formed somatic embryos on the epidermal layer without an
intervening callus phase (Fig. 2C). Excised regenerants subcultured on medium devoid of
PGRs formed whole plantlets (Fig. 2D).
Evidence of Indirect Shoot Organogenesis
Histological examinations of petiole sections harvested after 3 days of culture revealed
BAP-induced periclinal division in the subepiderrnal ce11 layers. After 3 to 7 days in
culture, the epidermal and subepidemal layers of the petiole explant started to divide (Fig.
3A). This callus tissue consisted of nurnerous meristematic zones [Fig. 3B (arrowheads)
and Cl. The cells of these promeristematic zones were srnaIl in size with dense cytoplasm
and prominent nuclei (Fig. 3C). Further differentiation of the promenstematic centers
formed meristematic zones that appeared as well defined dome-shaped shoot meristems by
day 21 (Fig. 3D). The shoot meristem developed leaf primordia and eventually forrned
shoot buds afler 22 to 33 days (Fig. 3E, arrowhead). A well-developed shoot bud
consisted of a dorne shaped shoot meristem surrounded by a few leaf primordia (Fig. 2F).
The leaf primordia had well developed trichornes (Fig. 3F, arrows). Vascular elements
(Fig. 3F, arrowhead) were observed sporadically dispersed within the callus, mainly near
the base of the shoot buds. By day 33 of the culture period, numerous de novo shoots were
visible on the surface of the petioIe (Fig. 2A and B). Shoots had an obvious comection to

the explant tissue and weI1-defined leaf primordia were also visible with the dissection
microscope.
Evidence of Direct Soma tic Em bryogenesz-s
In addition to shoot organogenesis, there was evidence of somatic embryogenesis in
both histological and visual observations of the petiole cultures. In histological
observations made at day 14, there were clearly visible penclinal and anticlinal ce11
divisions (Fig. 4A) and onset of organized development resembling proembryonic
structures was observed (Fig 4A, arrowheads). By day 33, late globular to early heart
shaped somatic embryos were clearly visible in histological sections (Fig. 4B). These
regenerants had a well-defined protoderm comprised of distinct rectangular cells and there
was no evidence of vascular connections to the materna1 vasculature (Fig. 4B). Late
cotyledonary stage sornatic embryos were also observed on the surface of the petiole
expIants exposed to BAP (Fig. SC). It is interesting to note the absence of a callus layer in
both the histological and morphological observations of those petiole sections that
regenerated via somatic embryogenesis (Fig. 2C, 4A and B).
Corzversiorz to Plantlets
Somatic embryos and shoots were separated fiorn the explant tissues and
subcultured on basal medium in test tubes for conversion to plantlets (Fig. 3D). More than
90% of al1 regenerants developed into intact plantlets within 30 days. Plantlets (90 -
100%) transplanted to a commercial soi1 mix survived and grew to maturity under
standard greenhouse conditions.

Figure 4. Histological evidence of somatic embryogenesis in petiole cultures of Echinacea
puvpurea L. in response to BAP.
A. Series of anticlinal and penclinal divisions leading to the formation of organized
proembryonic tissue by day 14 (arrowheads). Bar = 50 Fm. B. A well-developed hem-
shaped somatic embryo with a fuliy formed protoderm was observed at day 21. Bar-320
Pm-

Discussion
The value of commercial Echinacea production in Canada has been estimated to be
in the range of $25 million per annurn. The high value of this crop coupled with the limited
available uiformation about the unique biochemical processes in this plant species
warranted the development of protocols for the in vitro propagation of Echirzacea. The
most important results of this study are (1) the establishment of sterile seedlings in culture,
(2) the development of a protocol for in vitro regeneration from petiole explants and (3) the
simultaneous formation of shoots and somatic ernbryos in response to BAP.
Echinacea purpurea L. has been a difficult species to establish with conventional
tissue culture protocols. Systemic fûngal contamination was persistent and prolific.
Therefore, the elimination of contamination by incorporation of PPM in the seed
germination medium represented a major advancement in the development of culture
protocol. PPM is a broad spectrum biocide which targets enzymes in the citric acid cycle
and electron transport chah in bacteria and fungus (PPM, 1998). PPM does not transverse
ce11 walls of plants and therefore should not impede growth of plant tissues in culture
(PPM, 1998). However, in expenments with Echinacea purpurea, stunting of seedlings,
browning of tissues and reduced germination rates were observed at high levels of PPM.
These observations provide evidence of the need to optimize and minimize the use of PPM
for individual plant species.
In addition to the problems associated wiîh establishing sterile seedlings, Echinacea
purpurea L. was found to be largely recalcitrant to regeneration and unresponsive to many
plant growth regdators. In these studies, the cytokinin BAP was the most effective growth
regulator for Echinacea purpurea L. culture while the induction of regeneration by the

synthetic compound TDZ was limited. The regenerative Eequency increased when TDZ
and IAA were present in the induction medium. In many species TDZ was more effective
than BAP for the induction of shoots (Heutteman and Preece, 1993; Lu, 1993; Murthy et
al., 1998) and in several protocols, TDZ satisfied both the auxin and cytokinin requirement
for induction of somztic embryos (Visser et al., 1992; Murthy et al., 1998). Also, exposure
to TDZ in the culture medium stimulated the accumulation of endogenous auxins and
cytokinins (Murthy et al., 1995). The current observation that BAI? was more effective
than TDZ and that the action of TDZ can be modulated by exogenous auxin may be an
indication of the biochemical processes unique to Echinacea tissues.
A noteworthy observation of this study was indirect development of de novo shoots.
Indirect morphogenesis is defined as the formation of callus on explants and the subsequent
development of shoots or somatic embryos (Sharpe et al., 1980). Both histological and
morphological observations indicated that a callus layer formed initially on some of the
Echinacea purpurea L. petioles and that regenerated shoots onginated fiom within the
callus. As a result, vasculature linking the shoots to the explant tissue was only sporadically
visible in the undifferentiated mass of callus. In other petiole sections, the subepidermal
cells did not dedifferentiate to produce callus and somatic embryos were observed to form
on the epidermis.
The factors determining regenerative competence and the redirection of plant
growth and development remain largely undefined however it is apparent from recent
studies that nutritional, biochemical and environmental factors can determine the
developmental pathway of cornpetent cells. Skoog and Miller (1 957) hypothesized that the
route of morphogenesis was deterrnined by the relative ratio of auxins and cytokinins. As
*

well, researchers have hypothesized that auxin is required for the induction and cytokinin is
required for the expression of regeneration in plant tissues (Steward et al., 1964). In
cultures of Cicer arietinurn L., the expression of regeneration was shifted fiom shoot
organogenesis to somatic embryogenesis was induced by supplementation of the culture
medium with proline (Murthy et al., 1996a). The Echinacea purpurea L. regeneration
system developed in the current studies offers an unusual experïmental system for
investigation of the factors that favour sornatic embryogenesis since explants exposed to
the same culnire medium underwent different rnorphological processes. It is therefore
possible to speculate that different levels of endogenous biomolecules predetermined the
morphogenic potential of individual sections.
The micropropagation system for Echninacea purpurea L. developed in this
research will provide the basis for m e r investigations into the regdation of plant
morphogenesis, the quantification of the medicinally active biochemicals in Echinacea and
will provide technologies for the mass production of high-quality Echinacea prrrpzwea L.
for the commercial marketplace.

CHA PTER 4- REGENERA TION OF EC~ACEA PURPUREA HYPOCOTYL EXPLANTS V~il Srno T
ORGANOGENE~~S AND SOMA TIC EMBR YOGENESIS
Abstract
Shoot organogenesis was induced on two-week-oId etiolated hypocotyls of
Echinacea purpurea. Various auxins and cytokinins were compared in order to develop a
regeneration system including NAA, BAP and TDZ. Optimal regeneration was observed
in hypocotyls cultured on medium containing TDZ at high concentrations (20 p~-~-') for
short durations (3 days) or at low concentrations (0.5 p~-~-l) for a longer tirne penod (12
days). The cytokinin BAP also induced shoot organogenesis at concentrations of 5 and 10
p ~ - ~ for - a l period of 35 days. Somatic embryogenesis was induced via a callus phase
onginating koom Echinacea purpurea hypocotyl explants. Hypocotyl sections were
cultured on high concentrations of the auxin IBA to promote callus formation. The
callused explants were subsequently subcultured on the cytokinin BAP for the induction of
sornatic embryogenesis. The resulting regenerants were transferred to gib berelIic acid
(GA3) for fürther growth and proliferation. The procedure developed in this study provides
an effective method of mass producing clonally superior Echinacea purpurea plants.

Introduction
In the past decade plant tissue culture procedures have become usehl tools for the
horticultural industry. In vitro plant propagation methods developed allow for large-scale
micropropagation, which increases breeding efficacy and decreases the breeding cycle for
many crops (Altman & Ziv, 1997). This practice allows for the development of high
quality crops, which have been selected for horticulturally desirable traits for example
disease or pest resistance and stress adaptability (Altman & Ziv, 1997).
Somatic embryogenesis is also a valuable tool in plant tissue culture as regenerants
arise fiom a single cell, a high nurnber of somatic embryos may form on small amounts of
tissue representing a high proliferation rate, and conversion of somatic embryos to artificial
or synthetic seeds is possible (Mmhy & Saxena, 1998). As both cloning and genetic
modification are possible, somatic embryogenesis is a valuable method for crop
improvement through biotechnology (Sharpe et a1.,1980). Regeneration frorn callused
cultures on the other hand has been show to result in genomic variation in regenerants,
which are thought to arise during callus proliferation and the amount of variations may be
directly proportional to the duration culture period (Qureshi & Saxena, 1992). Although
this is not always desirable in some species, genetic variations rnay be a usehl tool in
improvement of medicinal plants as these changes may result in clones with higher levels
of valuable secondary metabolites.
An effective technique has previously been developed for the production of a shoot
organogenesis and somatic embryogenesis system utilizing Echinacea purpzrrea petiole
explants by exposure to a medium rontaining BAP only (Choffe et al., 2000). The
objectives of the current study was to develop an efficient micropropagation system for

Echinacea purpureu hypocotyl sections, preferably via somatic embryogenesis by
pnmarliy exposing the explants to altered ratios of auxins and cytokinins.
Materials and Methods
Establishmenr of Sterile SeedZings
Echinacea purpzrrea achenes were soaked overnight in a 10% solution of Plant
Preservation Mixture (PPM, 1998). The achenes were placed in a 250 ml flask. wrapped in
foi1 and placed in a growth room at 2S°C with continuous shaking at 150 rpm (G10 New
Brunswick Scientific Co. Inc., New Brunswick, NJ) for 16 hours. Echinacea achenes were
removed fiom the PPM solution and rinsed under running water for 10 min. The seeds
wer-e then surface sterilized by immersion in 70% ethanol for 30 sec, foIlowed by soaking
in a commercial bleach solution (5.4% sodium hypochloride) containing one drop of
Tween 20 per 500 ml for 18 min, and three rinses with sterile deionized water. Fifieen
disinfected seeds were cultured in 100 x 50 mm disposable Peti dishes (1Ydish)
containing 25 ml of culture medium which was comprised of !4 strength MS salts
(Murashige & Skoog, 1 962), !4 strength B5 vitamins (Garnborg et al., 1 968), 1 -5% sucrose,
and 2 ml-L-' PPM. The pH of the medium was adjusted to 5.7 with NaOK and HCI. The
medium was solidified with 0.3 % gelrite (Gellum gum, Schweitzerhall Inc., South
Plainfield, NJ) pnor to autoclaving at 1.4 kg.cm-2 for 20 min and dispensed into Petn
dishes (25 mlldish). The Petri dishes were sealed by wrapping twice in parafilm and placed
in a growth cabinet in complete darkness at 24OC for germination and seedling
development (Appendix 1).

Induction of Direcr Regeneration
Induction of regeneration was achieved by the addition of various plant growth
regulators to the media. Hypocotyl explants, L cm in length were cultured on a medium
containing MS salts (Murashige & Skoog, 1962), B5 vitamins (Gamborg et al., l968), 3%
sucrose and various plant growth regulators. The culture medium (pH 5.7) was solidified
with 3% gelrite added before autoclaving as previously described and dispersed in pre-
sterilized Petri 100 x 50 mm disposable Petri dishes (25 mvdish). The treatments
compared were NAA (0, 5 and 10 pmol-L-') and BAP (0, 5 and 10 pmol-~-') alone or in
combination, and TDZ (0. 0.5, 1, 2.5, 5, 10, 15 and 20 pmol-L") for a duration of 3, 6, 9
and 12 days.
Al1 cultures were incubated in a growth cabinet at 25°C under 16 hour photoperiod
(40-60 pmol-rn'2-s-') cool white fluorescent light (Phillips, Scarborough, Ontario).
Regeneration was quantified after 21 and 28 days for al1 cultures. Al1 treatrnents consisted
of 10 Petri dishes per treatment with 6 hypocotyl explants per dish and al1 experiments
were repeated at least twice. Statistical analysis was camied out using the Student
Newman-Kuells means separation test of SAS (Statistical Analysis System Inc., 1995).
Induction of Indirect Regenercztion
The induction of regeneration was achieved by the addition of various plant growth
regulators to the medium. Hypocotyl explants, 1 cm in length were cultured on MS salts
(Murashige & Skoog, 1962), B5 vitamins (Gamborg et al., 1968), 3% sucrose and IBA (50
and 100 pnol-L-'). The culture medium (pH 5.7) was solidified with 0.3% gelnte added
before autoclaving as previously described and dispersed in pre-sterilized Petri 100 x 50
mm disposable Petri dishes (25 ml/dish). Cultures were incubated in a growth cabinet at

2S°C under 16 hour photopenod (40-60 pmol-m-2-s-') cool white fluorescent light (Phillips,
Scarborough, Ontario) for a period of 4 weeks. Any roots which had formed from the
callus were excised and discarded and callused hypocotyls were then subcultured to plates
containing MS salts, B5 vitamins, 3% sucrose and BAP (2.5, 5, 7.5, 10 15 and 20 pmol-L-
1 ), which had been adjusted to a pH of 5.7 and solidified with 0.3% gelnte before
autoclaving. Al1 treatments consisted of 10 Petri dishes per treatment with 6 hypocotyi
explants per dish. Somatic embryos were excised fiom callused hypocotyls after 38 days
of culture and subcultured onto MS media containing 1.45pmol-~-' GA3 for plantlet
maturation and regeneration. Sornatic embryos were counted by hand and statistical
analysis was carried out using the Student Newman-Kuells means separation test of SAS
(Statistical Analysis System Inc., 1995).
Resttlts
Direct Shoot Organogenesis
The addition of TDZ to the media resulted in an overall swelling of the hypocotyl
tissues (Fig. 1). Quantitative observations indicated a significant decline in root
organogenesis with increasing TDZ concentrations (Fig. 2). TDZ effectively induced de
novo shoot organogenesis with the highest 6equency of regeneration at either 20 pnol-~-'
for 3 days, producing 3.45 shoots/explant, or 0.5 pnol-~-' for 12 days, producing 3.73
shoots/explant (Fig. 1 & 3). De novo shoot regeneration was also induced by the addition of
BAP to the culture medium with a maximal average of 2.81 shoots/hypocotyl at 5 Iimol-~'l
(Fig. 4). Supplementation of the BAP medium with the auxin NAA significantly reduced
the number of regenerants formed (Fig. 4), while NAA added to the media at al1

concentrations resulted in the formation of callus and completely inhibited root
organogenesis (Fig. 5).
Indirect Somatic Em bryogenesis
Echinacea hypocotyl explants cultured on high concentrations of the auxin IBA
produced green slightly compact callus formation (Fig 6). When subcultured onto basa1
medium or medium supplemented wiîh BAP, the morphoIogy of the callus remained
unchanged. At concentrations of BAP between 5 and 15 pnol-~-l the callus doubled in
size by day 33 and continued to expand. Callus cultured subcultured ont0 basal medium or
the medium containing low BAP concentrations aIso continued to produce roots, although
the formation of de novo roots declined as BAP concentrations increased (Fig. 7). Somatic
embryogenesis was apparent at al1 concentrations of BAP with the optimal treatrnents
being 4 weeks of culture on 50 pmol.~-l IBA and subculture on 2.5, 5, 7.5 and 10 pmol-~-l
BAP producing 6.0, 3.0, 3.2 and 4.1 roots/explant respectively, as well as the treatments of
100 pmol-~" IBA and 7.5 and 15 pmol-~-' BAP, which produced 6.9 and 5.7 roots/explant
(Fig 8). Al1 stages of somatic embryogenesis were obsewed as early as 2 weeks after
subculture ont0 B M (Fig. 9, 10 & Il) and this regeneration continued to increase up to
day 38 of subculture when-they were excised and transferred to GA3 for maturation.

Figure 1. Morphologie response of Echinacea puqurea hypocotyls to TDZ
A. Overall swelling of hypocotyl explants cultured on 5 pMT1 TDZ for 20 days afier 33
days of culture B. Shoot organogenesis from hypocotyl explants cultured on 20 @Id-'
TDZ for 3 days after 33 days of culture

Figure 2. Effect of the concentration and duration of the cytokinin TDZ on root
organogenesis of Echinacea purpzirea hypocotyls.
ab Values within a column with different superscnpts are significantly different (P

O 0.5 1 2.5 5 1 O 15 20
TDZ Concentration (pM)

Figure 3. Induction of shoot organogenesis on Echinacea purpurea hypocotyl explants in
response to different TDZ concentrations and induction period on TDZ-supplemented
medium.
abc Values within a column with different superscripts are significantly different (P

abc
O 0.5 1 2.5 5 10 15 20
TDZ Concentration (pIM)

Figure 4. Effects of the auxin NAA and the cytokinin BAP on shoot organogenesis
Echinacea purpurea hypocotyl explants after 33 days.
abdef Values within a column with different superscripts are significantly different (P

abc
Control SBAP 10BAP 5BAP-t IOBAI?+ SBAP+ IOBAI?+ SNAA 10N.A
SNAA SNAA IONAA IONAA
Growth Regulator Concentration (m

Figure 5. Effects of the auxin NAA and the cytokinin BAP on root organogenesis
Echinacea purpureu hypocotyl explants after 33 days.
abcd Values within a column with different superscripts are significantly different pcO.OS).

Control 5 BA. IO BAP 5BAP+ 5BAP + 10 BAP+ 10 BAP+ 5 NAA IONAA
SNAA 1ONAA SNAA 1ONAA
Growth Regulator Concentration (pM)

Figure 6. Callus formation on an Echinacea purpzlrea hypocotyl on 100 pmol-~-l IBA
after 4 weeks of culture.

Figure 7. Effects of the auxin B A and the cytokinin BAP on formation of root
organogenesis fkom callused Echinacea pzirpurea hypocotyl explants after 33 days.
abc Values within a colurnn with different superscripts are significantly different (Pe0-05).

50 50 50 50 50 50 50 100 100 100 100 100 100 100
IBA IBA IBA 5 IBA IBA IBA 1BA IBA IBA IBA 5 IBA IBA IBA IBA
2.5 BAP 7.5 10 15 20 2.5 BAP 7.5 10 15 20
BAP BAP BAP BAP BAP BAP BAP BAP BAP BAP
Treatrnent

Figure 8. Effects of the auxin B A and the cytokinin BAI? on formation of somatic
embryogenesis fiom callused Echinacea puqztrea hypocotyl explants afier 38 days.
abc Values within a column with different superscripts are significantly different (P

50 50 50 50 50 50 50 IO0 100 100 100 100 IO0 100
IBA IBA IBA 1BA IBA IBA IBA IBA IBA IBA IBA IBA IBA IBA
25 5 75 10 15 20 2.5 S 7.5 10 15 20
BAP BAP BAP BAP BAP BAP BAP BAP BAP BAP BAP BAP
Treatment

Figure 9. Different stages of somatic embryogenesis formed fkom callus induced by
culture of Echinacea hypocotyl explants on 100 pmol-L-l IBA and subcultured on 7.5
pnol-L-1 BAP after 38 days of subculture with BAP.

Figure 1 O. Globular stage somatic embryos formed fiom hypocotyl explants of Echinacea
purpurea L. cultured on 100 pnol-~" BA and 5 vmol-~-' BAP after 21 days of subculnire
with BAP.

Figure 1 1. Heart shaped somatic embryos formed kom hypocotyl explants of Echinacea
purpurea L. culhxed on 50 pmol-~" IBA and 2.5 pmol.~-l BAI? after 21 days of subculture
with BAP.

Discrtssion
As with Echinacea purpurea petiole explants, hypocotyls were also found to show
relatively Iow rates of shoot organogenesis when compared with other species. These
results confirm our earlier observation that is a relatively recalcitrant species, potentially as
a result of the unique complement of biornolecules. TDZ was found to be the most
effective PGR for promoting regeneration.
The cytokinin TDZ has previously been found to induce somatic ernbryogenesis
and shoot organogenesis in a range of recalcitrant species (Murthy et al., 1998; Lu, 1993).
The results of this study show that TDZ was effective for induction of shoot orsanogenesis
when the explants were exposed to hi& concentrations for a brief time period and also at
low concentrations for a longer period. In contrast, another cytokinin, BAP, was less
effective for induction of regeneration. These data indicate that the regenerative response
observed in the TDZ cultures arose fi-om additional factors to the cytokinin response and
that there exists an intimate interaction between concentration and time as a requirement
for induction. The mode of action of TDZ is not known but current findings indicate that
TDZ-induced morphogenesis arises fiom the modulation of endogenous gowth regulators
(Murthy et al., 1995; Hutchinson & Saxena., 1 W6),
The most significant finding of this study was the induction of callus and somatic
Embryogenesis by subsequent exposure of hypocotyls sections to auxin and cytokinin. It is
interesting in that this pattern of regeneration closely follow many valuable hypotheses as
to the mode of action of plant growth regdators. The ability of the callused expIants to
continue to forrn de novo roots suggests that the tissue rnay have become habituated to the
high auxin levels present in the media. Habituation is seen when tissues that normally

equire plant hormones such as auxins for growth suddenly lose this requirement and this
phenornenon has been reported for many species. In the case of habituation the cells lose
this endogenous auxin requirement but still retain the ability to produce roots. (Bennici &
Bruschi, 1999; Droual et al., 1998; Krikorian, 1995; Kevers et al., 1981). Another
hypothesis has been offered by De Klerk (1997) who suggested that unlike the auxin IAA,
which rapidly photo-oxidizes in culture media, B A might be stored for release when auxin
levels are low.
The formation of sornatic embryogenesis obtained when BAP was included in the
media may be a result of auxin being released and acting with the cytokinin to produce the
balance of auxidcytokinin necessary for regeneration (Skoog and Miller, 195 7). C yto kinin
concentrations included in the medium in inverse proportion to the BA exposure was
required thereby M e r supporting the concept that a delicate balance of auxinkytokinin is
required for somatic embryogenesis.
These observations as well as the continued growth and formation of sornatic
embryos will lead to some exciting future prospects. The prolific growth of the callus
suggests that continued subcultures of the callus will provide a useful tool in the Ion,- 0 term
micropropagation of Echinacea purpurea. It also offers a valuable starting point for this
method of in vitro propagation, which may be optimized by culturing explants on different
types and concentrations of auxins and cytokinins, varying pH levels, hormone levels and
other amendments to the culture media such as proline. In all cases, the mass-production of
clonally supenor plantlets of Echinacea purpurea may be obtained by the rnethods
achieved in this study.

C~PTER 5. DE NOvo ROOT ORGANOGENESIS IN HYPOCOTYL AND COTYLEDON CULTURES OF
ECHINA CEA PURPUREA L.
A bstra ct
An in vitro propagation system was developed for Echinacea purpurea L., a
medicinal plant cornrnonly used in the treatment of colds, flu and related ailrnents. The
effects of the auxins IAA, DA and NAA included in MS media at concentrations ranging
fiom 2.5 to 100 p ~.~-l were evaluated for their regeneration potentials. Root
organogenesis from Echinacea purpurea hypocotyl explants was effectively induced by
indolebutyric acid (BA) at concentrations ranging from 2.5 to 20 pM-L-'. Indoleacetic acid
(IAA) levels of 2.5 to 5 @PL-', as well as 15 to 20 p ~-~-', were optimal for root induction
and regeneration although this auxin was found to be less effective than BA, while
treatments with naphthaleneacetic acid (NAA) at dl levels (2.5 to 100 p ~-~-L) were
ineffective for induction of root organogenesis. Direct root organogenesis was induced
from IAA treatments at concentrations lower than 10 p ~ - ~ while - l concentrations behveen
10 and 100 I i ~ - ~ promoted - l root organogenesis fiom an intemediate callus stage. The
results of this sîudy have established a micropropagation systern for Echinncea pzirpztrea
that will provide stede plant material for further investigations into medicinally active
biochemicals and the mass production of high-quality Echinacea purpzrrea root tissues for
the commercial market.
introduction
The development of in vitro regeneration systems for medicinal plant species has
the potential to radically alter our approach to the production of plant-based medicines and
in vitro propagation allows for selection and clonal multiplication of genetically superior

individuals, which rnay facilitate the development of improved varîeties (Murch et al.,
2000). Protocols have recently been developed for regeneration of Echinacea plantlets via
somatic embryogenesis and de novo shoot organogenesis h-om petiole explants (Choffe et
al., 2000).
Since commercial preparations of Echinacea are comrnonly made korn root tissues,
in vitro protocols for root culture have the potential to improve commercial crop through
the production of sterile, consistent plant material in a shorter time frarne than is common
for field production. The development of micropropagation techniques for other
rnedicinally important pIant species vaIued for their root products have been successful
(Curtin, 1983). Ginseng is one such product in which numerous tissue culture techniques
have been developed. These techniques include somatic ernbryogenesis as well as well as
callus and ce11 suspension cultures (Wang et al., 1999; Shoyarna et al., 1995; Furuya et al.,
1986). The use of micropropagation techniques for Ginseng offer a more efficient way of
harvesting the active ginsenosides and polysaccharides produced by the species as the sarne
chernical constituents are produced by entire plants are also available in culture (Choi,
1988). Therefore the overall objective of this research was to evaluate the morphogenetic
capacity of hypocotyl and cotyledon explants of Echinacea pzqpztrea for root
organogenesis in response to various concentrations of the naturally occurring auxins l u
and B A and the synthetic auxin NAA.
Materials and Methods
Establishment of Siede SeedZings
Protocols for the establishment of sterile seedlings were identical to those described
previously (Chapter 4; Appendix 1).

Explant Culture
After 14 days explants of hypocotyls (each km) and cotyledons [Whole (intact), 5
mm, Distal (hrthest from the point of attachment to the hypocotyl) and Proximal (closest
to the point of attachent to the hypocotyl] were cultured on a medium consisting of MS
salts (Murashige & Skoog, 1962), B5 vitamins (Gamborg et al., 1968), 3% sucrose and
various plant growth regulators [IAA, IBA and NAA (0, 2.5, 5, 10, 15, 20, 50 and 100
prnol-~-']. The culture medium (pH 5.7) was solidified with 0.3% gelrite added before
autoclaving as previously described and dispersed in pre-sterilized Petri dishes (25
rnl/dish).
Al1 cultures were incubated in a growth cabinet at 25'C under 16 hour photoperiod
(40-60 pmol-m-2-d) cool white fluorescent light (Phillips, Scarborough, Ontario). Root
regeneration was quantified by visual observations afler 21 and 28 days for al1 cultures.
Al1 treatrnents consisted of 10 Petri dishes per treatrnent with either 6 hypocotyl explants or
3 whole and 6 sectioned cotyledon explants per dish and al1 experiments were repeated at
Ieast twice. Statistical analysis was carried out using the Student Newman-Kuells means
separation test of SAS (Statistical Analysis System Inc., 1995).
Results
Regeneration of Hypocovl Explants
The endogenous fûngal contamination of Echinacea seeds presented a difficult
problem in seedling establishment. PPM has been recently developed specifically for
tissue culture practices for the elimination of endogenous contamination of in vitro cultures
(PPM, 1998). In earlier experimentation the optimal concentration of PPM in the seed
semination medium was determined to be 3 ml-L-'. Higher concentrations of this mixture

were biostatic to Echhacea germination and lower amounts did not effectively eliminate
fimgal growth (Choffe et al., 2000). However, despite this amendment of the PPM, seed
germination was still less than 60%. It was subsequently found that the addition of MS salts
and B5 vitarnins to germination media allowed for >99% germination and the
establishment of healthy seedlings (Appendix 1).
14 day-old hypocotyls cultured on media devoid of growth regulators produced an
average of 0.5 - 2 roots/explant (Controls in Figs. 1, 2, 3, and 4). Supplementation of the
culture medium with NAA did not result in root organogenesis (Fig. 2) while both IAA and
IBA were fomd to effectively induce root formation (Figs. lB, C, and D). Rooting
response was induced by al1 auxin supplementations and the regenerated roots appeared
shorter and thicker than those in controls regardless of auxin type and concentration.
Significantly more roots were induced on hypocotyls exposed to between 5 and 20~rnol-~-'
BA (Figs. 3A and B). Root organogenesis fkom hypocotyls cultured on IBA at a11
concentrations was preceded witb an intermediate callus phase. There was a significant
increase in the number of roots between day 21 and 28 for both cultures exposed to IBA
and IAA (Figs. 3A & B and 4A & B). Hypocotyls exposed to high levels of IBA (100
pmol-~-') continued to express root organogenesis even after 3 - 4 weeks of subculture
ont0 MS basal media.
A different pattern of regeneration was observed on hypocotyls sections cultured on
the medium containing IAA (Figs. 4A and B). Significantly more roots formed on explants
cultured with 5 and 15-20 pmol.~-' of IAA than were observed for any other treatrnent.
Visual observations of the cultures indicated the potential for two distinct routes of root
formation. At lower concentrations roots formed directly fiom the cut ends of the explant

(Fig. 1C) but at higher concentrations an intermediate callus phase was obsewed (Figs. 1B
and D). Subsequent observations showed a distinct change in the developmental pathway
from direct to indirect differentiation at IAA concentrations of 9 - 10 pmol-~-' (Figure 5).
Regeneration of Cotyledon Explants
Root organogenesis was also induced on cotyledonary explants of Echinacea
seedlings. Root regeneration occurred both via an intermediate callus proliferation (Figs
6A and B) and directly fkom the cut ends (Fig. 6C). Cotyledon explants showed the greatest
response when cultured intact on 50 pnol.~-' BA producing an average of 9.7
rootslexplant, and as both proximal and distal explants cultured on 15 pmol-~'i IE!A
producing 9.1 rootdexplant (Fig. 7). included in the culture media at 10 pmol-~-i was
found to induce 6.6 rootslintact cotyledon (Fig. 8), which was highest for the IAA
treatments but significantly lower than IBA treatrnents.

Figure 1. Root organogenesis in cultured hypocotyl explants of Echinacea purpzwea L.
afier 28 days in culture.
A. Echinacea hypocotyl cultured on basal media. Bar = 30 mm. B. Indirect root
organogenesis on Echinacea hypocotyl explants on 5 pnol-~-' IBA. Note the initiation of
roots £Yom callus. Bar = 18 mm. C. Direct root organogenesis fiom cut ends of hypocotyl
explant on 5 pmol-~-l IAA. Bar = 18 mm. D. Indirect root organogenesis on hypocotyl
explanting the presence of 15 pnol-L" LAA. Note the development of callus. Bar = 5mm.

Figure 2. Root organogenesis of Echinacea purpurea hypocotyl explants cultured on NAA
after 28 days in culture.
ab Values with a colurnn with different superscnpts are significantly different (P

O 2.5 5 7.5 I O 12.5 15
NAA Concentration (pM)

Figure 3. Root organogenesis of Echinacea purpurea hypocotyl explants cultured on EA.
A. Effect of the auxin IBA on root organogenesis of Echinacea purpurea hypocotyl
explants after 21 days of culture. B. Effect of the auxin B A on root organogenesis of
Echinacea pulpurea hypocotyl explants after 28 days of culture.
abc Values within a column with different superscnpts are significantly different (Pc0.05).

O 2.5 5 IO 15 20 50 100
IBA Concentration (pw
O 2.5 5 IO 15 20 50 100
IBA Concentration (PM)

Figure 4. Root organogenesis of Echinacea purpurea hypocotyl explants cultured on IAA.
A. Effect of the auxin IAA on root organogenesis of Echinacea purpurea hypocotyl
explants after 21 days of culture. B. Effect of the auxin IAA on root organogenesis of
Echinacea purpurea hypoco tyl exp lants afier 28 days of culture.
abc Values within a column with different superscripts are significantly different (P

O 2.5 5 10 15 20 50 100
IAA Concentration (CLM)
IAA Concentration (FM)

Figure 5. Effect of IAA concentration on root ongin (direct or indirect regeneration) in
Echinacea purpurea hypocotyl explants after 28 days in culture.

5 6 7 8 9 10 11 12 23 14 15
IAA Concentration (IcM)
Route of Root
Organogenesis
Direct
Indirect

Figure 6. Root organogenesis in cultured cotyledon explants of Echinacea purpzirea L.
after 28 days in culture.
A. Indirect root organogenesis fiom an intact Echinacea cotyledon explant on 50 pmol-~-l
BA. Bar = 9 mm. B. Indirect root organogenesis from a distal section of an Echinacea
cotyledon explant on 15 pmol-~-L IBA. Bar = 15 mm. C. Indirect root organogenesis from
a proximal section of an Echinacea cotyledon explant on 15 pmol-~-l IBA. Bar = 9 mm.

Figure 7. Effects of the auxin B A on root organogenesis of Echinacea purpurea cotyledon
explants after 28 days of culture.
ab Values within a column with different superscripts are significantly different (P

IBA Concentration (CLM)

Figure 8. Effects of the auxin IAA on root organogenesis of Echinacea puvpzrrea
cotyledon explants after 28 days of culture.
abc Values within a column with different superscripts are significantly different (Pc0.05).

5 -
4 -
3 -
2 - abc
abc
abc abc
abc abc
abc
abc
O 2-5 5 10 IS 20 50 100
IAA Concentration (@f)
. Whole
Distal

Discussion
The most significant results of this study are development of in vitro protocols for
the induction of root organogenesis from both hypocotyl and cotyledon explants of
Echinacea exposed to the naturally occu~ng auxins IAA and [BA. The auxin BA was
found to be the most effective for induction of root organogenesis while the synthetic auxin
NAA was completely ineffective. At low concentrations of IAA (5 pnol.~-') rooting was
observed at the cut ends of the hypocotyl explants and at higher concentrations (20 pmo1.L-
1 ) through an intermediate callus phase. At intermediate concentrations of IAA, the root
formation was significantly decreased. Closer observation of root organogenesis showed a
decline in the amount of roots forming directly fiom the epidermis between 9 and 10
,urnol.~-' MA and an increase in rooting fiom callus at the same concentrations. This
observation shows the change in the developmental pathway from direct to indirect
differentiation of Echtnacea purpurea hypocotyls occurs within these concentrations.
Cotyledon explants of geranium have been shown to be highly responsive to
various hormonal treatments (Murthy et al., 1996b). Cells contained in the cotyledon
region are highly regenerative have been shown to exhibit both root and shoot organogenic
activity in culture (Murthy et al., 1995; Obeidy & Smith, 1993). The formation of a large
number of root organogenesis fiom Echinacea cotyledon explants cultured on IAA and
BA has also exhibited the regenerative ability and responsiveness of these explants to
auxin treatments. While removal of proximal sections of cotyledon explants of geranium
cultures was detrimenta1 in that it inhibited regeneration (Murthy et al., 1996b), the culture
of cotyledon explants into distal or proximal sections of Echinacea did not affect root
organogenesis. This observation along with the ability of these srna11 explant sections to

develop a higher amount of roots than the larger hypocotyl sections will provide a means to
optimize the micropropagation system of Echinacea.
In general and in these expenments, callus is formed at the highest levels of
exposure to auxins (Skoog & Miller, 1957). It is interesting to note that roots were formed
on sections exposed to M and B A but only callus wos observed on the sections exposed
to NAA at any level. While differences in the mode of action of the three auxins are not
known, Our results indicate significant differences in auxin strength in the promotion of
root organogenesis. From this data it appears that the efficiency of auxins for root
organogenesis is IBA>IAA>NAA.
In preliminary experiments, Echinacea explants continued to grow when transferred
to liquid culture. The development of a micropropagation procedure to produce roots fiom
hypocotyl tissue may then be expanded to include proliferation in a bioreactor system.
This system may then be utilized to produce large amounts of stenle root tissues for
analysis, purification, extraction and standardization of their active ingredients.

CHAPTER 6: I?ESI/LTS AND FUTURE PROSPECTS
The overall objective of this thesis was to develop a micropropagation system for
Echinacea purpurea. an increasingly valuable crop in the Canadian and worId herbal
markets. Plant material for Echinacea products are normally collected fiom the wild or
from field grown plants, ar,d problems with this practice include: a) contamination of the
plant matenal with insects, hngi and bacteria, b) the length of time required for production
of a saleable product, c) plant-to-plant and year-to-year variability in the active components
of the plant matenal and d) the lack of pure standardized plant material for analysis.
Micropropagation is a valuable tool for the medicinal plant industry, as it allows for the
production of sterile, consistent plant material for biochernical characterization and
commercial production.
The most significant findings of the thesis were:
I. Echinacea purpurea petioles exposed to the cytokinin BAP h e d both somatic
embryos and shoots.
3. Hypocotyl explants of Echinacea pulpurea formed de novo shoots when cultured
on a medium containing the plant growth regdator TDZ at high concentrations for
short penods of tirne or low concentrations for longer time penods.
3. Hypocotyl explants of Echinacea purpurea cultured ont0 IBA for 4 weeks were
induced to fom callus. Subculture of the calli on a medium containing BAP
resulted in somatic embryogenesis.
4. Echinacea purpurea root organogenesis was induced on hypocotyl and cotyledon
explants cultured on a media supplemented with either the auxin BA, or the auxin
IAA. The optimal concentration of IBA was between 5 and 15 j ~~-l-'.

The results of this research program provide new evidence of a difference in
morphogenic response of various plant tissues to the same inductive stimulus.
Organogenesis and sornatic embryogenesis were both induced on Echinacea petiole
explants cultured on the cytokinin BAP only. These findings indicate that it is possible to
have different morphogenic responses kom the same type of tissue explant exposed to the
sarne plant growth regulator. In contrast, hypocotyl explants exposed to cytokinins
produced de novo shoots but pretreatrnent of the same tissue with an auxin led to a two-
stage regenerative response. The initial exposure to auxin induced callus formation and
exposure of the callus to cytokinin resulted in somatic embryogenesis. Previous
researchers have found evidence that both explant source and plant growth regulators were
crucial for the expression of morphogenesis (Steward et aI., 1964; Skoog & Miller, 1957).
These findings confirm the earlier hypothesis but fiuther indicate that the endogenous
metabolites of the tissues cm affect the morphogenetic cornpetence.
These findings provide new opportunities for both the study of rnedicinal plant
physiology and for the medicinal plants industry. The regeneration systems developed for
Echinacea purpurea may be used as model systems for the establishment of protocols for
study of the unique physiology of medicinal plants. Together, these expenments have
established optimized protocols for the mas-production of Echinacea. Medicinal plant
species produce a variety of active secondary metabolites, and the availability of sterile
plant matena1 is the first prerequisite for biochemical characterization. By utiIizing the in
vitro techniques developed for Echinacea as a model system, researchers will have a tool
for the evaluation of the active ingredients of this species as well as for other medicinal
plant species producing similar arrays of chemical compounds.

In addition, the ability to mass produce Echinacea purpurea in sterile culture
environments provides new technologies for the medicina1 plant industry. In vitro culture
will allow for the production of consistent products. For example, it is now possible to
produce Iarge quantities of root tissues in a bioreactor. Another application of these
technologies may be the selection and mass, clona1 propagation of superior individuals
within the Echinacea population. With these techniques it is possible to identiQ and select
lines of Echinacea purpurea with higher leveIs of medicinal compounds or resistance to
disease.

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APPEND~XI: THE DE VELOPMENT OF STERiLlZLi TïûN PROCEDURES FOR ECHINACEA PURPURE.4 L.
ACHENES
Echinacea purpurea seeds used throughout this thesis showed an extremelly high degee of
endogenous fimgal contamination. Sterilization procedures rnost comrnonly used in for
plant tissue culture proved inadequate for Echinacea and thus more stnngent techniques
were developed The development occurred through numerous steps, which ultimately
resulted in approximately 99% contamination Bee cultures. The steps leading to the
successful generation of contamination free seed cultures are laid out in the following tabIe:

Table 1 : Sterilization methods and results for Echinacea purpurea achenes.
Sterilriation Process
1. 30 sec dip in 70% ethanol
18 min ernmersion in 5.4% sodium
hypochIoride
solution containing one drop of Tween 20 per
500 mi
3 rinses in sterile disti11ed water
Seeds cultured on water agar (8 g-l-')
2. Seed coat removed
Seeds stedized and cultured as in step #1
3. Seeds sterilized as in step #1.
Seeds cultured on water agar (8 g-~-')
supplemented with 1, 2, 3, 4 or 5 ml-1-' PPM
4. Seeds soaked in full strength PPM for
15 and 30 minutes
50% of seeds were then stedized as in
#1 and 50% were plated directly
Each treatment cultures containing:
- water agar containing O or 0.5 ml-1-'
PPM
- !4 strength MS media containing O
or 0.5 ml-1-' PPM
5. Seeds treated as in step #5 with
treatments also including cultures
containing full strength MS media
containing O or 0.5 ml-1-' PPM
6. Repeated step #5 soaking instead for 30
and 60 minutes
1 7. Seeds soaked in full strength PPM for
30, 60 minutes and overnight
Seeds sterilized as in step #I
Seeds plated ont0 the following
FeatmentS:
water agar, !A or full strength MS media
supplemented with O or 0.5 ml-1-' PPM
8. Seeds soaked in fi111 strenght PPM for 1
hour or 2 hours and in a 5% PPM
solution overnight
Seeds are sterilized as in step $1
Seeds cultured on !4 or hl1 strength MS media
supplemented with 0, 0.5 or 1 ml-1-' PPM
9. Seeds soaked in 5, 10 and 20% PPM
solution overnight
Seeds are sterilized as in step #1
Seeds cultured on '/z strength MS media
1 supplernented with ml-1.' PPM
-
Results
100% contamination
100% contamination
[PPM]3 ml-1- Higher percentage of clean plates.
Auch browning and stuÏ-iting of explants
Al1 seeds plated directly (without sterilîzation) were
stunted and brown
Optimal conditions resulted in 50% contamination
free cultures
Results as for step #4
Results as for step M
Resuits as for step #4
Much less contamination and healthier growth was
observed in treatrnents consisting of overnight soak
in PPM and plates containing % strength MS media
supplemented with 1 ml-1-' PPM
Overnight soaks in a 10% PPM solution followed
by the treatrnent described resulted in virmally 99%
contamination free plates and healthy growth of
Echinacen purpurea seedlings

APPENDIXE T m s PUBLEA T/ONS
Choffe, K.C., Victor, J.M.R., Murch, S.J. & Saxena, P.K. 2000. In vitro regeneration of
Echinacea purpureu L.: Direct somatic embryogenesis and indirect shoot
organogenesis in petiole culture. In Vitro Cell Dev. Biol-Plant. 36(1):30-36.
Choffe, K.C., Murch, S.J. & Saxena, P.K. 2000. Regeneration of Echinacea purprrrea L.:
Induction of root organogenesis from hypocotyl and coty1edon explants. Plant Ce11
Tiss. Org. Cult. (In Press).