TISSUE CULTURE OF WESTERN HEMLOCK DWARF MISTLETOE AND ITS APPLICATION TO STUDIES ON BIOLOGICAL CONTROL Shannon J. Deeks B.Sc. (with Distinction), University of Victoria, 1995 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Biological Sciences O Shannon J. Deeks 2000 SIMON FRASER ONIVERSITY MI rights remcd This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author.
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TISSUE CULTURE OF WESTERN HEMLOCK DWARF
MISTLETOE AND ITS APPLICATION TO STUDIES ON
BIOLOGICAL CONTROL
Shannon J. Deeks
B.Sc. (with Distinction), University of Victoria, 1995
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in the Department
of
Biological Sciences
O Shannon J. Deeks 2000
SIMON FRASER ONIVERSITY
MI rights r e m c d This work may not be reproduced in whole or in part, by photocopy
or other means, without permission of the author.
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ABSTRACT
Dwarf mistletoes (Arceuthobiurn spp.) are parasitic flowering plants that attack
commercially valuable conifers. The biology of this genus along with 22 other geneni of
parasitic flowering plants that have been cultured in vitro is reviewed in detail with
respect to distribution, host range. and tissue culture procedures. A procedure for in vitro
culture of western hemlock dwarfmistletoe (A. tsugense Rosend. G.N. Jones subsp.
tsugenre) is described. A factorial experiment evaluated the effects of media,
temperatures, presence or absence of light, and plant growth regulators on the production
of radicles, holdfasts, and callus. Optimal conditions for radicle elongation were White's
medium at 20°C in the presence of light and without plant growth regulators. Holdfasts
developed fkom tips of radicles or fiom swollen radicles, and were maximally produced
with Harvey's medium, iight, 2,4-dichlorophenoxyacetic acid (2,4-D) at 1 mg 1-', and 6-
benzy laminopurine (B AP) at 0.1 mg 1-' . Callus arose fiom split radicles and hold fasts
and optimal development was on White's medium with 0.5 mg 1-' 2,4-D and 1 mg T'
BAP at 20°C in the dark. The tissue culture procedure was used for in vitro screening of
calli and germinated seeds of A. tsugense subsp. tsugense with potential fungal biological
control agents (Cy findrocurpon cylindroides and Colletohichum gloeosporioides).
Mistletoe tissues were prepared for light microscopy 1,2,3,7 and 14 days post-contact
with fiingi. The process of pathogenesis was investigated, and specimens were rated for
extent of colonization. Cushion development, ce11 wall degradation, and both
intercellular and intracellular colonization were evident for germinated seeds challenged
with both fûngi. Gmwth of dwarf mistietoe callus was reduced by both fun@, and ce11
iii
wall degradation dong with intercellular and intracellular colonization was evident. Cells
infected with Cylindrocarpon cyfindroides were disorganized and appeared plasmolysed.
The in vitro screening method was useful to elucidate the host-pathogen interactions, and
sensitive enough to determine that Cylindrocarpon cylindroides was more aggressive at
colonization than Colletohichum gloeosporioides.
1 would like to thank the following people for making this thesis a redity: Dr.
Simon Shamoun of the Pacific Forestry Centre for his advice, encouragement, and
patience, Dr. Zarnir hinja for his excellent reviews and for securing b d i n g to send me
to Montreal for the CPS meeting, and Dr. Patrick von Aderkas of the University of
Victoria for his fiiendship and encouragement. 1 also wish to thank Ed Wass for
assistance with seed collections, Hugh Barclay for statistical advice, and Lesley Manning
and Terry Holmes for microtechnique assistance. I also gratefully acknowledge the
funding provided by Forest Renewal B.C., and the Natural Sciences and Engineering
Research Cowicil of Canada.
I also wish to thank my lab mates, Tod Ramsfield and Carmen Oleskevich, for
their constant support during my studies. Finally, 1 thank Mom, Dad, Missy, Peeps,
Wendy, and Von for their endless encouragement.
TABLE OF CONTENTS
. . APPROVAL ............................................................................................................... 11
... ................................................................................................................... AB STMCT 111
............................................................................................. ACKNOWLEDGMENTS v
.............................................................. ............................. TABLE OF CONTENTS .... vi
LIST OF TABLES ........................................................................................................ ix
........................................................................................................ LIST OF FIGURES x
CHAPTER 1 .................................................................................................................. 1
...................................................................................................... 1 . iNTRODUCTION 1
1 . 0. INTRODUCTION .............................................. ........................................ 1
........................................................................ 1.1 . General information 1
..................................................................... 1 .2 . Hosts and distribution 1
1.4. Biology and life cycle ..................................................................... 3
1 . 5. Detection ..................................................................................... 5
............................................................................. 1 .6 . Control methods 6
1 . 7. Tissue culture. ................................................................... 9 . . . .........................................*.**.......**.................. 1 8. Researc h objectives 10
.................................................................................................................. CHAPTER 2 1 1
2 . TISSUE CULTURE OF PARASITIC FLOWERING PLANTS: METHODS AND
............................... APPLICATIONS IN AGRICULTURE AND FORESTRY .. ....... 1 1
........................................................................................ 2.0 INTRODUCTION 1 1
2.1 TISSUE CULTURE: METHODS AND RESULTS .................................. 16
.................................... .........*....................... 2.1 . 1 Convolvulaceae ... 16
................................................................................. 2.1.3 Loranthaceae 18
2.1.4 ûrobanchaceae ........................................................................ 20
2.1 .5 . Santalaceae ................................................................................... 24
2.1.6. Scrophulariaceae ......................................................................... 2 8
2.1.7. Viscaceae ................................................................................. 37
... 2.2. APPLICATIONS OF TISSUE CULTURE: PRESENT AND FUTURE 4 1
CHAPTER 3 ............................................................................................................... 4 7
3 . IrN VITRO GERMINATION AND DEVELOPMENT OF WESTERN HEMLOCK
DWARF MISTLETOE (ARCEUTHOBIUIM TSUGENSE SUBSP . TSUGENSE .......... 47
3.0. INTRODUCTION ..................................................................................... 47
3.1. MATERIALS AND METHODS ................................................................ 50
3.1 . 1 . Seed collection ............................ .., .......................................... 5 0
3.1 .2 . Pre-saeening seeds for viability .................................................. 50
3.1.3. Media ........................................................................................... 51
3.1.4. Experimental design ................................................................. 51
3.1 .5 . Light rnicroscopy ........................................................................ 52
3.1.6. Statistical analysis ........................................................................ 52
3.2. RESULTS AND DISCUSSION ................................................................ 53
3.2.1. Radicle development .................................................................... 53
3.2.2. Holdfast development ............................................................... 55
3.2.3. Callus development ............. ... .................................................. 61
3.3. CONCLUSIONS ....................................................................................... 65
.................................................................................................................. CHAPTE2 4 67
A HISTOPATHOLOGICAL STUDY OF INFECTION OF GERMINATED SEEDS
AND CALLUS OF WESTERN HEMLOCK DWARF MISTLETOE BY
CYLIhllROCARPON CYLI1VDROLUE.S AND COLLETOTRICHOM
GLOEOSPORIOLDES IN DUAL CULTURE .............................................................. 67
...................................................................................... 4.0. WIRODUCTION 6 7
4.1. MATERIALS AND METHODS ............................................................... 68
4.1.1. Mistletoe tissue ............................................. ....................... 6 8
4.1.2. Fungal growth .............................................................................. 68
4 . I . 3. Dual culture ................................................................................. 6 9
4.2. RESULTS ................................................................................................... 72
.............................................................................. 4.2.1 . Fungal growth 72
.................................................................................. 4.2.2. Dual culture 72
4.3. DISCUSSION ............................................................................................. 83
5.0. CONCLUSIONS AND FUTURE RESEARCH ..................................................... 87
6.0. REFERENCES .................................................................................................... 90
viii
LIST OF TABLES
Table 1 . Classification of parasitic flowering plants that have been cultured in viîro.. 13
Table 2. Tissue culture media used for experiments on parasitic plants ....................... 25
Table 3. Responses of 23 genera of parasitic plants to tissue culture conditions. ........ 30
Table 4. Colonization of germinated seeds of dwarf mistletoe challenged with Cylindrocatpon and Colletohichunr as expressed by number of hyphae in 1 mm2 of
............................................................................................................................. tissue. 76
Table 5. Colonization of callus of dwarf mistletoe challenged with Cylindrocarpon and Colletotrichum as expressed by ratings (0-5) and number of hyphae in 1 mm2 of tissue (in
................................................................................................................... parentheses). 82
LIST OF FIGURES
Figure 1 . Schematic representation of parasite seed germination and parasite-host .......................................................... interactions for seven families of parasitic plants 14
Figure 2 . Potential responses of parasitic plants to tissue culture conditions ............... 15
Figure 3 . Arceuthobiun mgense seed cultures on Harvey's medium .......................... 39
Figure 4 . Steps in germination of mistletoe seeds and production of dicles. holdfasts ................................................ and callus on Harvey's medium and White's medium 5 6
Figure 5 . Holdfast production on Harvey's medium and White's medium at 20°C ..... 58
Figure 6 . Influence of plant growth regulators on holdfast production ......................... 59
Figure 7 . Light micrograph of a spherical holdfast produced nom a swollen radicle on Harvey's medium &er 7 months in culture at 20°C light (1 mg 1-' 2. 4.D and 1 mg f '
............................................................................................................................... BAP) 60
Figure 8 . Callus production for al1 treatments based on percentage of seeds that produced ......................................................................... callus fkom split radicles or holdfasts 6 2
Figure 9 . Light micrographs of a spiit radicle with callus .......................................... 6 4
Figure 10 . Schematic division of germinated seed of dwarî mistletoe showing areas used ................................................................... in ratings of colonization by the two fùngi 7 1
Figim 1 1 . Average fimgal colony diameters (mm) d e r 2 weeks for A) Cylindrocatpon and B) Colletohichum on various media, with standard error bars ............................... 73
Figure 12 . Endosperm parenchyma cells infected by Cylindrocarpon ......................... 74
Figure 13 . Dwarf mistletoe gexminated seeds infected by Colletotrichum ................... 77
................................ Figure 14 . Dwarf mistletoe radicles infected by Cylindrocarpon 7 8
Figure 15 . Growth of callus in the absence of (control. represented by open circles) and ................ presence of (dual culture. represented by closed circles) of Cylindrocapon 79
Figure 16 . Growth of calius ia the absence of (control. represented by open circles) and prrsence of (dual culture. represented by closed circles) of Colletotrichum ................. 80
Figure 17 . Callus that has been wlonized by Cylindrocarpon ..................................... 81
CEIAPTER 1
INTRODUCTION
1.0. INTRODUCTION
1.1. Genet al information
Dwarf mistletoes (Arceuthobium spp.) are widespread and destructive parasitic
flowering plants attacking Pinaceae and Cupressaceae in North and Central America,
Afiica, Asia, Europe, and the West Indies. There are 42 species of dwaxf mistletoe,
attacking a wide range of conifers including western hernlock, lodgepole pine, ponderosa
pine, Douglas-fit, tme fin, western larch, spnices, and many others (Tattar 1 989).
Unusual feanires of dwarf mistletoe include explosive seed dispersal, formation of
witches' brooms (abnormally profuse, dense mass of host branches), seeds with
chlorophyllous endospenn, and radicles with stomata (Hawksworth and Wiens 1996;
Sinclair et al. 1987).
1.2. Hosts and distribution
There are 34 species of dwarf mistletoe in North America, 5 of which are found in
Canada. Eastern dwarf mistletoe (A. pusillum) is a parasite of black spruce (Piceu
mariuna (Mill.) BSP), white spruce (Picea g h c a (Moench) Voss, and red spruce (Piceu
rubens Sarg.). It is the only Canadian species not present in British Columbia (BC),
occUmng in Hudson Bay, the Cumberland areas in eastan Saskatchewan to southern
Manitoba, southem Ontario, Qwbec, and the Maritime Provinces including
Newfoundland. There are 4 species in BC. Lodgepole pine dwarf mistletoe (A.
americanum Nutt. ex Engelm.) attacks lodgepole pine (Pinus contorta Dougl. ex Loud.
var. latfoliu Engelm.) in the interior of BC. Douglas-fir dwarfmistletoe (A. douglasii
Engelm.) attac ks Douglas- fir (Pseudo tsuga menziesii (Mirb. ) Franco var. menriesii) in the
extreme southem interior. Larch dwarf mistletoe (A. laricis (Piper) S t. John) attacks
westem larch (Lani occidentalis Nutt.) in the southeasteni interior, and hemlock dwarf
mistletoe (A. tmgense (Rosend.) G.N. Jones) attacks hemlock on the coast. Hemlock
dwarf mistletoe is divided into two subspecies; western hemlock dwarfmistletoe (A.
tsugense subsp. tmgense), which occurs on western hemlock (Tsuga heterophylla (Raf.)
Sarg.), and mountain hemlock dwadmistletoe (A. mgense subsp. mertensiunae), which
occurs on mountain hernlock (Tsuga mertensiana (Bong.) Cm.). Two different races of
western hemlock dwarf rnistletoe have been descriied (Hawksworth and Wiens 1996).
The western hemlock race attacks western hemlock and the shore pine race attacks shore
pine (Pinus conforta Dougl. ex Loud. var. contorta). A. tmgense subsp. tsugense is found
near the North American West coast of Canada (prirnarily in British Columbia) and the
United States (Alaska, Washington, Oregon, and Califomia)(Hawksworth and Wiens
1996). The principal host of A. mgense subsp. mgense is western hemlock, secondary
host is amabilis fi, and occasional or rare hosts include sitka spruce, grand fir, mountain
hemlock, and western white pine (Unger 1992).
13. Damage
Arceuthobium is the single most destructive pathogen of commercially valuable
timber ûes in Canada, western United States, Mexico, and parts of Asia (Hawksworth
and Wiens 1996). Substantial annual volume losses to conifers in BC is atûibuted to the
impact of dwarf mistletoe. Dwarfmistletoes weaken trees by slowly removing water,
minerais, and photosynthates (Hawksworth and Johnson 1993), causing reductions in
vigour, growth, seed production, wood quality, and even host mortality (Kirnmey and
Graham 1 960; Haw ksworth and Wiens 1 996). Dead tissues produced b y dwarf rnistletoes
provide enû=y points for stain and decay funpi (Hawksworth and Wiens 1996). Wood
quality is reduced via witches' brooms, branch swellings and stem infections with the end
result being abnormal grain, min impregnation, spongy wood texture, and increased
number and size of b o t s (Hawksworth and Wiens 1996). Infected wood has subnomal
strength and poor pulping quaiity due to abnormally short, distorted tracheids and a high
proportion of ray tissue (Sinclair et al. 1987). Dead branches and trees in stands damaged
by dwarfmistletoe can becorne fire hazards (Sinclair et al. 1987). in addition, dwarf
mistletoe weakens, disfigures, and kills landscape trees in tesidentid areas and parks
(Sinclair et al. 1987).
1.4. Biology and life cycle
Generally, dwarfmistletoe has a five year life cycle. In the au- of the first
year (early October for western hemlock dwarf mistletoe), seeds are explosively
discharged due to buildup of water pressure in the seed (Hawksworth and Wiens 1996).
Seeds can travel at a rate of 80 km per hour and can land up to 16 meters away fiom the
source tree (Hawksworth and Wiens 1996). At the time of seed discharge, viscin cells
(long filamentous cells attached to the endocarp) rupture and release a sticky mucilage
(Mscin) which enables the seed to stick to objects they land on (Scharpf 1970). During
the first min, viscin cells absorb water and swell. The slippery seeds slide down the
needles and adhere to the twig, becoming f i d y attached to the host upon drymg, and
dried viscin cells also protect the endosperm and embryo, thus reducing water loss
(Scharpf 1970). Atter a period of overwintering on the twig, the seeds germinate
(Febniary for western hemlock dwarfmistletoe). Germination refers to the emergence of
the radicle tkom the seed. When the radicle reaches an obstruction such as a bark crack or
base of a needle, needle bundle or bud (Baranyay and Smith 1972), a holdfast (smooth
swelling) forms at the tip of the radicle. At this time, a haustonal wedge arises fiom the
center of the holdfast and penetrates the host, and the endophytic system which is made
up of a network of branching filaments, is subsequently formed. With establishment of
dwarf mistietoe in the host, the seed and holdfast eventuaily di:: and disintegrate
(Baranyay and Smith 1972), leaving the endophytic system intact. The endophytic systern
consists of cortical strands and sinkers. Cortical strands are structures that ramifiy
throughout the i ~ e r bark of the host, and sinkers are structures that orighate from
cortical strands and grow toward the cambium where they become embedded in
successive layers of xylem (Baranyay et al. 197 1 ; Alosi and Calvin 1984). In the second
year, host cells increase in size (hypertrophy) and number (hyperplasia) and a swelling
becornes visible at the infection site (Unger 1992). AAer the endophytic system is well
established, it produces buds fiom which aerial shoots develop in the third year (Agios
1997). Aerial shoots produce branches either in a fan-like (flabellate) fashion (A.
tsugense, A. Zaricis, A. douglasii) or a whorled (verticillate) fashion (A. americanum).
Male shoots are referred to as starninate, whereas fernale shoots are pistillate. in the
fourth year, flowers are produced on aerial shoots, and pollination (via insects or whd)
and fertilization occurs. Male and fernale flowers are produced on separate plants, but
both sexes may grow on the same host tree or even on the same branch (Kimmey and
Graham 1960). Male flowers have 3-4 petal-like paris, pollen sacs, and nectaries.
Femaie fiowers are closed and inconspicous, and produce stigmatic exudate to attract
pollinators. The fifth year witnesses h i t maturation and seed dispersal, thus completing
the life cycle.
1.5. Detection
Detection involves recognizing the symptoms and signs of dwarf mistletoe
infection. S ymptoms of infection include spindle-shaped swellings, cankers, and witches '
brooms (Baranyay et al. 197 1 ; Agrios 1997). Signs of infection include aerial shoots
appearing at the infection's advancing edge, and basal cups. Basal cups are the cup-like
remnants on the bark of an infeftion which remains visible long &er the disintegration of
aerial shoots (Baranyay et al. 197 1 ). Hemlock dwarf mistletoe causes localized infections
(anisophasic) that are restricted to swellings whereas lodgepole pine and Douglas-fi
dwarf mistletoe cause systemic infections (isophasic) in which the infection keeps pace
with the terminal buds of the new growth (Kuijt 1960). There is no obvious swelling in
systemically infected branches, but aerial shoots are produced in a regular fashion dong
the branches (Baranyay and Smith 1972). Most recently, a polymerase chah reaction
(PCR) method was developed to detect early dwarhistletoe Section in Douglas-fir and
western larch (Marler et al. 1999).
1.6. Control methods
There are four control methods for dwarf mistletoe: 1 ) silvicultural, 2) chemical,
3) genetic resistance, and 4) biological control with h g i and insects. Dwarfrnistletoe is
amenable to silvicultural control due to its slow spread, confinement to above-ground
parts of their hosts, host specificity, dependence on living trees, and easy detection (Kuijt
1955; Hawksworth and Wiens 1996). Silvicul~iral control methods include clear-cuts,
elimination of residual trees, sanitation thinning in young infected stands, the use of
mistletoe resistant species, and the use of barriers (natural or artificial), such as planting
resistant trees in a 20 m sûip next to clear-cuts in areas of dwarfmistletoe infestation
(Hawksworth and Wiens 1996). Pruning off infected branches cari improve the health of
the tree and lengthen its life (Hawksworth and Johnson 1993). As the new foresty code in
BC calls for small cut-blocks and selective harvesting rather than clear-cuts, the ratio of
edge trees to seedlings increases and this results in conditions ideal to dwarf mistletoe
spread, intensification, and establishment (Sharnoun 1997).
The use of chernicals is another control option for dwarfmistletoe. ~ the~hon@
( ~ l o r e l ~ , cepham, ~threl@) is an ethylene releasing agent and is the only chemical
approved by the Environmental Protection Agency in the United States (Hawksworth and
Wiens 1996). This chemical, 2-chloroethylphosphonic acid, causes shoot abscission, thus
delaying seed production for 2-4 years. It has also been found to cause partial dieback of
dwarf mistletoe brooms (Livingston and Brenner 1983). Ethephon was able to stimulate
abscission of eastem dwarfmistletoe (A. pusihm) aerial shoots on black spnice (Picea
mariana)(Livingston and Brenner 1983; Livingston et al 1985). The major disadvantage
of ethephon is that it does not kill the endophytic system and respmutbg wiil occur
(Adams et ai 1993). In addition, aerial application is prohibited, thus this control method
is time-consurning, and there is no promise of longtenn control (Hawksworth and Wiens
1 996).
There has been occasional evidence of genetic resistance in several host-parasite
combinations. Resistance mechanisms operate at various stages in the life cycle, ranging
fkom a reduction of seed germination on some hosts to apparent immunity (Smith 1974).
AAer germination, resistance may take the form of a low percentage of infection, whereas
afier infection, there may be a low rate of endophytic growth or a sparse production or
complete absence of aerial shoots (Smith 1974). Other signs of resistance are swellings
with a low length to width ratio, and the absence of even minor host branch proliferation
or broorning (Smith 1974). As the seeds of dwarf mistletoe are coated with viscin cells
which absorb water, the slippery seeds tend to slide down the needles to the needle base
where they establish infection on the branch. Roth (1966) noted that ponderosa pine,
with its characteristic drooping, horizontal needles, seemed to be resistant, and seeds slide
down the horizontal needles onto the ground instead of sliding down the needle to
establish infection on the branch. However, when scions with drooping needles were
grafted to healthy rootstock and were outplanted on test sites, this drooping phenotype
was lost. Roth ( 1974) found that physiological factors may be operating in ponderosa
pine as he found host necrosis in tissue beneath the holdfast mggesting incornpatibility.
Retardation of germination of A. laricis. A. omericunum, and A. douglasii seeds on spruce
was sufficiently consistent over 4 years to point to an inhibitory substance or substances
in needle or bark tissue (Smith 1974). Scharpf (1987) noted one seed source of JeBey
pine in California (Placer seed source) that was particuiady resistant to dwarf mistietoe.
Resistance was characterized by a reduced nurnber of infected trees, infections per tree,
and infection intensity. Scharpf (1987) found this resistance to be heritable and
outplantings were made in California carnpgrounds. Smith et al. (1993) noted that there
was evidence of resistance mechanisms operating within the host branches of western
hemlock infected with dwarf mistletoe. Scions of resistant îrees were grafted ont0
healthy rootstock and then rarnets of successful grafts were grown in pots and later
inoculated with seeds. Resistance mechanisms persisted. Resistance seerned to result
fiom the reduced ability of the penetrating structure to enter the host cortex or early
demise of the structure before infection. Snyder et al. (1 996) looked at monoterpenes in
oleoresin of xylem and phloem and found that they could tell parasitized from non-
parasitized trees based on phloem chernistry, suggesting that host chernicals, specifically
monoterpenes, may be involved with genetic resistance.
Biological control with fun@ is another potential control method for this
destructive parasitic plant. There are 29 kwwn fungai parasites of dwarfrnistletoe and
18 of these are found in Canada. Nectria neornacrospora (anamorph Cylindrocarpon
cyfindroides) is a canker fungus that attacks swellings caused by dwarf mistletoe,
resulting in death of rnistletoe-infected branches (Hawksworth and Wiens 1996). It is
attractive as a biocontrol agent because it causes rapid canker production (Funk et al.
1973), reduces aerial shoot production without damaging the host tree (Smith and Funk
1980), and produces an abundant number of spores (Funk et al. 1973). By colonizing the
endophytk systern, Nechia neomacrospora fan reduce the production and p w t h of
aaiai shoots (Smith and Funk 1980). Colletotrichum gloeosporioides is the most lethal
and widespread pathogen of Arceuthobiuni, causing shoot dieback. (Hawksworth and
Wiens 1996). This fungus is attractive as a biocontrol agent, as it is destructive to the
host, it sporulates abundantiy on liquid medium, and the spores germinate and grow over
a wide temperature range (Wicker and Shaw 1968). Cylindrocurpon gillii foms lesions
on stems and causes anthracnose and shoot dieback (Hawkswoith and Wiens 1996). It is
capable of capable of producing abundant inoculum in culture (Wicker and Shaw 1968).
Hyperparasitic fhgi can be used singly or in combination to control the survival,
reproduction, and impact of dwarf rnistletoe.
1.7. Tissue culture
Tissue culture is the culture of isolated explants on artificial media. This field
encompasses a great diversity of culture methods, including embryo, organ, protoplast,
and suspension cultures in addition to excised tissues (Bonga and von Aderkas 1992).
Studies on in vitro culture of dwarf mistletoe have been rare. Blakely (1959) attempted to
grow A. doughsii in vitm. Although his efforts to infect Douglas-fir callus cultures with
seeds were unsuccessful, the endophytic system of the parasite when present in cultured
tissue proceeded to invade the callus. A. purilium, a parasite of black spruce, has been
grown in vitro, and radicles (branched, unbranched), callus, embryoids, and seedlings
with holdfasts and haustonal wedges were f o d in culture (Bonga 1965; Bonga 1968;
Bonga and Chababorty 1968; Bonga 1969; Bonga 197 1 ; Bonga 1974). Most recently,
Deeks et al. (1 997; 1998; 1 999) have mccessfully cultured A. tsugense subsp. tsugense,
witnessing the production of radicles (branched, unbranched), callus, and seedlings with
holdfasts.
1.8. Research objectives
The first objective of this research was to comprehensively review the 23 genera
of parasitic fiowering plants belonging to 7 families containhg parasitic plants and
highlight relevant information on biology, distribution, host range, tissue culture
procedures, and the applications of tissue culture techniques to study the biology and
host-pathogen interactions of these parasites. Since western hemlock dwarf mistletoe (A.
lsugense subsp. tsugense) hm not previously been grown in tissue culture, the second
objective was to determine optimal conditions for growth, germination, and production of
callus and the early seedling stage. The third phase of research involved the discovery of
a suitable medium for dual challenge studies using dwarfmistletoe and two potential
biological control fiinpi (Cylindrocarpon cylindroides and Colleioîrichum
gloeosporioides). Germinated seeds and callus were then challenged with these fûngi in
vitro to determine if there was any muhial influence (stimulation, inhibition) between the
b g i and the dwarf mistletoe. The colonized explants were examined histologically in an
effort to detemine the mechanism of b g a l infection.
CHAPTER 2
TISSUE CULTURE OF PARASITIC nowEmG PLANTS:
METHODS AND APPLICATIONS IN AGRICULTURE AND
FORESTRY
2.0 INTRODUCTION
There are over 3000 species of parasitic flowering plants distributed arnong 1 7
families (Press et al. 1990). Of these, nearly 40 species in 23 genera from 7 different
families have been cultureci in vitro (Table 1). The major characteristic of these plants is
that during a part of their life cycle, they depend on the host plant for some or al1 of their
nutrients. Two types of parasitic relationships exist: holopmitic (obligate parasites) and
hemiparasitic (facultative parasites). Holoparasites are totally dependent on their hosts
for nutrition, since they possess no chlorophyll or capacity to assirnilate carbon and
inorgimic niîrogen. Hemiparasites are not totally nutritionally dependent on their hosts,
since they possess chlorophyll but require water, minerals and physical support £tom their
hosts. These plants can parasitize stems or roots (Stewart and Press, 1990; Marvier 1996)
and their life cycle is completed by the production of flowers and seeds. The stages in
parasite developrnent fiom sead germination to vascular connection with the host are
schematically presented in Fig. L.
During parasite seed germination, a radicle emerges whose tip swells to produce a
haustorium (holdfast in mistfetoes) which penetrates the host. The haustorium is a naturd
and specialized morphological and physiological bridge that is comprised, at least in part,
of living tissue and through which nutrients and water are transported fiom the host to the
parasite (Kuijt 1969). The hausto~urn therefore functions for attachment, penetration,
and watedsolute acquisition (Kuijt 1969; Press and Graves 1995; Stewart and Press
1990). The penetration structure, termed the endophyte or sucker, is initiated from the
distal, central part of the haustorium (Press and Graves 1995). Pnmary (pre, upper)
haustoria are formed on the radicle whereas secondary haustoria are fomed on any organ
other than the radicle (Parker and Riches 1993).
In vitro culture of parasitic plants is important for many reasons. Morphological
studies can be done on the endophytic system, various host-parasite relationships
(mechanism, physiology, biochemistry, signaMeceptors) can be examined, and
physiological studies leading to the control of the more destructive parasitic plants can be
investigated. Secondary metabolite production (i.e. cancer dnigs) can be studied, along
with the micropropagation and genetic irnprovement of plants with commercial value.
In tissue culture, explants taken fiom parasitic plants may produce callus, shoots,
mots, seedlings, somatic embryos, haustoria, and floral buds (Fig. 2). Explants or callus
have beeen used to derive ce11 suspensions and protoplasts. Regenerated plants can be
obtained from somatic embryos or shoots developing h m callus (Fig. 2). The various
tissue culture media that have been utilized are summarized in Table 2. The results
obtained are presented in Table 3.
Table 1. Classification of parasitic flowering plants that have been cultured in vi*oa
Convolvulaçeae Cucuta arvensis, campesrris, chinensis, Sympetalae japnica, reflexa, rrifolii
Lauraceac Cassytha filiformis Archichhydeae
Loranthaceae Am-vema rniguelii, pendulum. quanàang Archichlamydcae
Amylofheca dicryophleba Archichlamydeae
Nuytsia jloribunda Archichlamydcae
Scurmla philippensis. pulverulenta Archichlamyk
Tapinanthus bangwensis Archichlarnydeae
Oro banchriccae Aeginetia indica S ~ m w a e
Cisranche ntbulosa Sympctalae
Orobanche oegyp tiaca Sympetalae
Santalaceae
Osyris wighriana Archichlamydtac
Santalurn acumitumm, album, Archichlamydeat lanceolatum. spicaturn
Scrophulariaccae Agalinis purpureu SYrnWw
A lecrra orobanchoides. sessi/lora. S ympetal ac vogelii
Melumpytum lineare S Y r n W a e
Sopubia delp hinifo lia S Y r n ~ l a e
Striga crsiarica, euphrurioides, SYrnpctaI~ gesnerioides, hennonthica
Arceurhobium douglarii, pusillum. tsugense Archichlarnydtac
Phoradendmn fluvescens, tomentosum Arc hic hlamydeae
Yiscwll album Archichlamydeae
a Al1 belong to the Class Dicotyledoneae.
Tubiflorae
b a i e s
Santaiales
Santalales
Santalales
Santalales
Santalales
Santalales
Santalales
Tubiflorae
Tubiflorae
Tubi florac
Santalales
Santal aies
Santalales
Tubiflorae
Tubiflorae
Tubiflarat
Tubi florae
Tubiflorae
Sanialales
Santaldes
Santalales
Convolvulineae
Magnoliineae
Loranthineae
Loranthineae
Loranthineae
Loranthineae
Loranthincae
Loranthineae
Lomthintae
Solanineae
SoIanineae
Solanineac
Santalincae
Smtalinerie
SantaIineae
Solanineae
Solanineae
Solanineae
Solanineae
Solanuieac
Loranthineae
Loranthineae
Loranthineac
Figure 1 . Schematic representation o f parasite seed germination and parasite-host interactions for seven families of parasitic plants.
Regmemted Plants
Doriot phnta
Figure 2. Potential reponses of parasitic plants to tissue culture conditions.
2.1 TISSUE CULTURE: METHODS AND RESULTS
In this review, the general biology of 23 genera of parasitic plants belonging to 7
families is considered and their responses to tissue culture are described (Table 1). This
is followed by a discussion on the applications of tissue culture techniques to study the
biology and host-pathogen interactions of these parasites. The potential areas for future
research are highlighted.
2.1.1 Couvolvulaceae
Cuscuta (dodder). Cuscuta is a holoparasitic stem parasite which is herbaceous,
viny and rootless and has scale leaves, clusters of small flowers, and mound-like
haustoria (Parker and Riches 1993). It is mainly distributed in warm temperate and sub-
tropical areas, most commonly in North America, the Mediterranean, the Middle East,
and South Asia (Press and Graves 1995). Dodder affects growth and yield of a range of
plants, including aifalfa, flax, clover, tobacco, onion, canot, potato, sugar beet, soybean,
coffee, citnis, and omamentals (Press and Graves 1995), causing slight loss to complete
crop destruction (Agrios 1997).
In tissue culture, the physiology of flowering was studied (Tanaka et al. 1995) and
it was determined that Cuscuta is a qualitative short day plant (Tanaka et al. 1995).
Flowering depended on the adantiauxin ratio in the medium. A low concentration of
indoleacetic acid (IAA) eriiianced flowerhg, while a high concentration inhibited it, and
addition of TIBA (an antiauxin) pnnnoted f l o w e ~ g (Swamy and Baldev 1988).
Shoot tip culture of Cuscuta was used to study the hormonal regulation of lateral
bud development by the apex. Since leaves or roots are absent fkom the shoot tip, it
provided a simpler system for study than the whole plant (Maheshwari and Sreekrishna
1982; Rajagopal et al. 1988). Application of IAA to the shoot tip explant delayed lateral
bud development (Maheshwari and Sreekrishna 1982). Excision of the apical bud from
the shoot tip resulted in growth of a shoot fiom the lateral bud (Maheshwari and
Sreekrishna 1982), while addition of gibberellic acid restored apical dominance
(inhibition of lateral buds) since it caused an overproduction of auxin at the apex
(Maheshwari et ai. 1980; Maheshwari and Sreekrishna 1982). Stem segments of Cusnrta
were used to study auxin transport, since their simple morphology, the absence of
secondary sites of auxin synthesis eg. leaves, and the long zone of elongation provided
suitable study material (Maheshwari and Sreeknshna 1982). It was found that the
capacity to transport auxin basipetally increased dong the length of the vine and was
related to the growth potential of the apex (Maheshwari and Sreekrishna 1982). The in
vivo patterns of straight and coiling growth of Cuscuta was mimicked in vitro, and high
auin and cytokinin levels induced straight growth (to form vines) while low levels
induced coiling (to twine around the host). Tight coiling was associated with numerous
haustoria (Rajagopal et al. 1988).
Cassytha (laurel). Cassythu is a holoparasitic, viny stem parasite of many
angiosperm species in India, with s d e leaves and mound-lilce haustoria which penetmte
into the vascular system of the host. Embryo germination, seedling development and
radicle development were observeci in tissue culture (Rangan and Ranga Swamy 1969).
Seedlings develo ped fiom embryos placed on modi fied White's medium lacking plant
growth regulators, but a primary root was absent and adventitious roots fonned instead.
The plumule developed nonnally into a shoot bearing scale leaves (Rangan and Ranga
Swamy 1969).
2.1.3 Loranthaceae
Most mistletoes (largely shoot haniparasites) occur in tropical and subtropical
climates worldwide and attack hardwood forest and shade trees, gymnosperms (juniper,
cypress) as well as cofEee, cacao, rubber, apple, cherry and citnis (Agrios 1997). Although
mistletoes are very destructive, littie is known about their growth, physiology, or aspects
of the host-parasite relationship (Bajaj 1970). During seed germination, a radicle emerges
which attaches to the host, and produces a pad (holdfast) (Fig. 1) from which the
endophyte tbat penetrates the host is produced. The plumule (embryonic shoot) emerges
fiom in between the two cotyledons (Bajaj 1970). The dependency of the parasite on the
host stimulus for seed germination and the chexnical factors initiating haustorium
formation were studied in tissue culture (Bhatnagar 1987).
Amyema, Amylotheca. These two genera are Australian mistletoes that d u c e
growth and cause mortality of their host, eucalyptus (Yan and Reid 1995). The effects of
hormones and nutneats on Amyema leaf development and morphology were studied using
d u s and seedings grown on modified Murashige and Skoog medium containhg L M or
NAA. Seedlings fonned plumular leaves and haustonal discs in vitro (Hall et al. 1987).
Seedlings with holdfasts, haustotial discs and plumular leaves developed in Amylotheca
kom mature embryos on White's medium with casein hydrolysate and iAA (Johri and
Bajaj 1964; Bajaj 1970).
Dendrophthoe. Dendrophthoe is a stem hemiparasite on teak, mango, citms,
custard apple, eucalyptus, apple, peach and guava in India (Parker and Riches 1993). The
nutritional requirernents for parasite growth and induction of polyembryony (John and
Bajaj 1965) and factors promoting ernbryo development were studied to elucidate the
phy siology and nature of the host-parasite relationship (Bajaj 1 968). Undifferentiated and
embryogenic callus, embryoids, buds (shoot, floral) and seedlings with holdfasts and
haustorial discs developed on White's medium. Haustonal formation was induced by
adjusting the ratio of cytokinin to auxin, and high cytokinin (low auxin) resulted in shoots
and low cytokinin @gh auxin) resulted in haustoria development (Ram and Singh 199 1 ).
Nuytsia. Nuytsia is the largest of al1 mistletoes and is found in Western Australia
where it is called "Christmas Tree" because of the orange flowers produced around
Christmas-tirne. It is a root parasite of grasses and cultivated carrots (Kuijt 1969) and is
considered a primitive parasite, since the large embryo, which is completely enclosed in
the endospenn, has fiee, well-developed cotyledons that develop into the h t foliage
leaves. Other primitive features include a root cap, a shoot apex that can give rise to a
shoot, and the absence of terminal haustoria. Shoots, roots, embryogenic callus and
somatic embryos were produced on White's medium with casein hydrolysate, BA, and
kinetin (Nag and Jobri 1969; Nag and Ram 1977).
Scumla. Scumla is a stem parasite found on several hosis including conifers,
citnis, peach and guava in India and South and East Asia (Parker and Riches 1993).
Callus, shoots, seedlings with haustoria, and somatic embryos were produced on White's
medium with casein hydrolysate and IAA (Bhojwani and Johri 1970).
Tapinunthus. Tapinunthus is a herniparasite of citrus, peach, papaya, mango,
avocado, shea butter nut, rubber, teak, almond and pecan in Afkica, Ethiopia and Southern
Nigeria (Parker and Riches 1993). In T. bangwensis, a parasite of cocoa in Ghana,
haustorial development was studied in tissue culture. Callus, shoots and seedlings were
produced on White's medium, with embryo germination and holdfast formation occurring
without a host (Onofeghara 1972).
Taxillus. Tuxi11u.s is a parasite of crop species in Africa and Asia (Parker and
Riches 1 993). In both T. vestirirs and T. cuneafus, factors affecting organogenesis were
studied (Nag and John 1976). Development of shoot buds and haustoria was influenced
by growth regulator combinations (Johri and Nag 1970; Nag and Johri 1976).
2.1.4 Orobanchaceae
Mernbers of this family (broomrapes) are root holoparasites that attack herbaceous
crop plants includllig potato, tomato, tobacco, alfalfa, clover, and hemp (Agrios 1997).
They are widely disiributeci in Europe, the United States, Afnca, and Asia (Agrios 1997).
Brwmrapes have a fleshy stem bearing a simple spike of flowers and small scales instead
of leaves (Parker and Riches 1993).
Aeginetia. Aeginetia is a parasite that attacks sugarcane, nce, maize and sorghum
in India, China, Japan, the Philippines and Lndonesia (Parker and Riches 1993).
Germination usually requires host root exudates and involves enlargement of epidermal
cells near the micropylar end of the seed but not the emergence of a radicle or root. A
nodule is thus fomed from which tendnls are produced in response to the host seedling
root. The tendrils may help facilitate the connection with the host (Kato and Hisano
1983). The mechanism controlling tendril formation and identification of tendnl-
inducing stimuli were studied in tissue culture. Tendril formation was found to be
stimulated by host root (Misconthus) extract (Kato and Hisano 1983). Ce11 division
(septation) of the tendril was promoted by sucrose, glucose or cytokinin and longitudinal
ce11 division by cytokinin (Kato et ai. 1984).
Cistanche. Cistanche, a parasite on crownflower, madar and acacia, was culhired
in vitro to study the requirements for germination and growth and development of the
embryo (Rangan 1965). Callus and shoots of C. tubulosa were produced in the absence
of a host (Rangan 1965; Ranga Swamy 1967). In this species, dong with Orobanche,
seed germination was induced by coconut milk or casein hydrolysate and it was suggested
that these nutrients replaced the host stimulus (Rangan 1965).
Orobanche (broomrape). Orobanche is a parasite that reduces yields of field and
vegetable crops, including lettuce, cabbage, mustard, rape seed, saftiower, sunflower,
cucumber, melon, squash, eggplant, potato, tobacco, tomato, faba bean and lentil (Parker
and Riches 1993) in regions of Asia, the Mediterranean, and the Middle East
(Musselman 1980; hess and Graves 1995). Embryos are globular at seed maturity and
lack demarcation intc radicle, plumule and cotyledons (Ranga Swamy 1963). In nature,
seeds genninate after a conditioning period of appropriate moisture and temperature
regimes (Van Hezewij 1993). A germ-tube emerges fiom the radicle end of the embryo
and forms a primary haustorium and a tubercle (nodule), on which a shoot bud
differentiates, evenhially elongating and ernerging to produce the shoot (Fig. 1).
Nurnerous root-like organs arise nom the tubercle and infect host roots via secondary
haustona. Upon contact with the host mot, sticky papillae are fomed which adhere to the
root surface. Penetration of the host root is facilitated by enzymatic action, resulting in
separation of the host cells (Parker and Riches 1993). in nature, parasite shoot growth
and seedling development required nuûients fiom the host (Rangan 1965). In fact, seeds
can be dormant for 12 years until contact is established with the host root or the host
stimulus is provided (Worsham et al. 1959; Parker and Riches 1993). The chernical
acting as the nahual germination stimulant has been partially purified but not identified
(Parker and Riches 1993).
In tissue culture, the mechanisms of penetration by the haustorium into the host
root and the signals controlling haustorial initiation, host attachent, and directional
growth were studied (Ben-bod et al. 199 1 b). In an experirnental system, tomato roots
were aseptically exposed to Orobanche calli. It was observed that callus protrusions
behaved similarly to radicles emerging from germinating seeds and once contact occwed
with the host root, a haustorium-like structure was fomed which developed into a
tubercle. Anatomical examination reveaied a normal fusion between parasite xylem and
the vascular system of the host root (Ben-hod et al. 199 1 b). Another experirnental system
involved studying intact host plants infected with parasite seedlings under aseptic
conditions (Losna-Goshen et al. 1996). In a divided peûi dish, one side contained a
medium that supporteci growth of the host (tomato) root, while the other side contairieci a
nutrient-poor medium that sustained growth of Orobanche. Once the host root grew over
the divider, it stimulated germination of Orobanche seeds, and was infected by
Orobanche seedlings.
The role of pectin methylesterase (PME) produceù in infective calli and seedlings
of Orobanche was recently investigated in vitro (Ben-hod et al. 1993; Losner-Goshen et
al. 1998). The PME softened the host tissue by degrading the middle larnella, facilitating
parasite penetration of the host. Direct evidence for the presence of PME in the infection
zone of the haustoriurn in situ was obtained from cytochernical and irnmunocytochemical
studies (Losner-Goshen et al. 1998). PME was detecteù in the cytoplasm and cell walls
of Orobanche intrusive cells and in adjacent host apoplast. A second enzyme,
polygalacturonase (PG), involved in pectin dissolution, was identified in callus of O.
aegvptiaca (Ben-hod et al. 1997), but its fiuiction during infection is unknown. It was
postulated that PG degraded wall pectins and loosened cells to facilitate penetration of the
parasite into the host cells (Losner-Goshen et al. 1998).
ui 0. aegvptiaca, a parasite of Brassica campestris, coconut milk and casein
hydrolysate replaced the host stimulus and induced se& germination, while watermelon
juice induced shoot differentiation in the absence of any host root stimulus (Ranga
Swamy 1963). Shoots foxmed on differentiating calli originating at the radicular end of
the ernbryo (Ranga Swamy 1963; Rangan 1965) on complex, undefined media. It was
postulated that the reqwement for complex media was related to the undifferentiated
status of the ernbryo (Oko~kwo 1975).
2.1 .S. Santalaceae
Most members of this f d y are hemi-parasitic, root-infecting herbs and shrubs,
except for the sandal tree and Exocarpos cupressiformis, an arborescent parasite (John
and Bhojwani 1 965). The latter is ofien called "cherry tree" due to its red and green h i t
(Kuijt 1969). Shoot buds were regenerated from endosperm c d u s of fiocarpos on
modified White's medium with IAA and kinetin (Johri and Bhojwani 1965).
Osyrjs. Osyrs is a shrubby sandalwood substitute that produced callus from
endosperm on modified White's medium with 2,4D and casein hydrolysate (Johri and
Bhojwani 1965). When the medium was supplemented with iAA and kinetin, instead of
callusing the endosperm produced outgrowths which showed patches of vascular tissue
( J o b and Bhojwani 1965).
Santalum. The sandal tree (S. album) is native to India and Indonesia and is
planted in South America, Australia, Hawaii, New Zealand and in other regions. Its
highly vaiued fiagrant wood is used for carvings, furnihue, idols, and craftmanship
articles, and its oil is used for cosmetics, soap, perfume, incense, and medicine (Rao and
Bapat 1992). It is not surprising that another name for this tree is '%agant gold". The
mua1 world requirement for sandalwood oil is 200 tonnes, which is equivalent to 10,000
tonnes of wood, and only 10% of this demand can be met From natural resources
(Lakshmi Sita 199 1).
Clonal propagation of sandalwood in tissue culture was attempted for several
reasons. The tree is difficult to propagate by vegetative means, such as grafbg and
rooting cuttings, and trees over 3 years of age are prone to spike disease, a destructive
Table 2. Tissue culture media used for experiments on parasitic plants
MediWn B5 (Gamborg 1975)
Basal medium (individual authors)
C 1 (Wei and Xu 1990)
Hoagiand's solution (Hoagland and Amon 1938)
Harvey's medium (Harvey 1967)
K medium (MS salts+BS vitamins) (Muras hige and Skoog 1962; Gamborg 1975)
Knop's medium (Knop 1865)
Linsmaier and Skoog medium (Linsmaier and Skoog 1965)
Modifed Munishige and Skoog medium (individual authors)
Modified Hoagland's solution (Loo 1945)
Modified White's medium (individual authors)
Parasitic plant Striga asiatica (Worsham et al. 1959; Okonkwo 199 1; Wolf and Timko 1991; Cai et al. 1993)
Cuscuta campestris (Loo 1946), C. reflexa (Maheshwari and Sreeknshna 1982), Melampynirn lineare (Cantlon et al. 1963)' Santalum album (Bapat and Rao 1979; Rao and Bapat 1980; Bapat and Rao 1984)' Sopubia delphinijofia (Shivanna and Ranga Swamy 1976)' Striga asiatica (Williams 1961)
Striga asiaticn (Cai et al. 1993)
Cuscuta campestris ( L w 1946)
Arceuthobium pusillum (Bonga 1969; Bonga 1 97 1 ), A. tsugense (De& et ai. 1997)
Cuscuta anensis (Binding 1974), C. reflexa (B inding et al. 1 98 1 ), C. tnyofii (Bakos et al. 1995)
Alectra vogelii (Okonkwo 1979, Cusczrta japonica (Furuhashi 199 I ; Furuhashi et ai. 1995; Tanaka et al. 1999, Phoradendronflovescens (Cdvin 1966)
Srnga asiatica (Cai et al. 1993)
Amyema miquelii (Hd et al. 1987)' A. pendulum (Hall et al. 1987), A. quandang (Hall et ai. 1987)
Cuscuta campestris (Loo 1946)
Alectra vogelii (Okonkwo 1979, Amyerna pendulum (Johri and Bajaj 1964), Amylotheca dicryophleba (John and Bajaj 1964; Bajaj 1970), Arceuthobium pusiflum (EBonga 1968; Bonga and Chakraborty 1968; Bonga 1969), Cc~ssythhafilifomis (Rangan and Ranga Swamy 1969)' Cistanche ntbulosa (Rangan 1965; b g a n and Ranga Swamy 1968), Curcuta reflaa (Baldev 1959a; Baldev 1959b; Maheshwari and Baldev 196 1; Mafieshwari et al. 1980)' Dendrophrhoe jülcata (Bajaj 1966; Bajaj 1970; Bajaj 1995; John and Bajaj 1962; Johri and Bajaj 1963; Iohn and Bajaj 1965; Iohri and Nag 1968; Rarn and Nag 1986; Ram and Nag l988), Exocurpos cupress#iormiS (Bhojwani 1969)' N w i a froribunda ( h g and Johri 1969; Nag and Johri 1976), Orobanche aegyptiaca (Ranga Swamy 1963), O@ wightiana (John and Bhojwani 1965)' SontaIum album
Medium
Murashige and Skoog medium (Murashige and Skoog 1962)
Nitsch's medium (Nitsch 195 1)
Potato dextrose agar (individual authors)
Tepfer's medium (Tepfer et al. 1963)
V47 medium (Binding 1974)
Water agar (inâividual authors)
White's medium (White 1943)
White's minerai solution (Bakos et al. 1995)
pulvemlenta (Bhojwani and Johri 1970). usiatica (Cai et al. 1993), S. euphrasioides (Ranga Swamy and Rangan l966), S. gesnerioides (Okonkwo 1982), Tapinanthw bungwenF1î (Onofeghara 1972), Taxillus cuneaw (Nag and Johri 1976), T. vestiîus (John and Nag 1970; Nag and Johri 1976)
Agalind purpurea (Riopel and Musselman 1979; Steffens et ai. 1982). Alectra orobanchoides (Trautmann and Visser 1987), Alectra sess&flora (Okonkwo 1992), A. vogelii (Okonkwo 1975; Trautmann and Visser 1987)' Orobanche aegypîiaca (Ben-hod et al. 1 99 1 a; B en-hod et al. 199 1 b), Phoradendron tomentosum (Bajaj 1970). Santafum anrrninatum (Barlass et al. 1980). S. album (Laksbmi Sita l98Oa; Rao and Raghava Ram 198 3; Bapat et al. 1985; Rao and Ozias-Akins 1985; Bapat and Rao 1988; Bapat et al. 1990; Lakshmi Sita 1991; Valluri et al. 199 1; Rao and Bapat 1992; Bapat et al. 1996; Rugkhla and Jones 1998), S. lanceolatum (Barlass et al. 1980), S. spicatm (Rugkhia and Jones 1998); Shiga asiatica (Chidley and Drennan 1987; Okonkwo 199 1; Wolf and Timko 1992; Babiker et ai. 1994), S. gesnerioides (Okonkwo 1982), S. hermonthica (Okonkwo 1966a; Okonkwo l966b; Okonkwo 1970), Vircum album (Becker and Schwarz 197 la; Becker and Schwarz 197 1 b; Fukui et al. 1990)
Phoradendron/lavescenî (Calvin 1 966), Sopubia delphinifolia ( S h i v a ~ a and Ranga Swamy 1976)
Striga asiatica (Okonkwo 1992)
Chtanche tubufosa (Rangan 1965; Rangan and Ranga Swamy 1969)
Santalum album (Bapat et al. 1985; Rao and Ozias-Akins 1985; Rao and Bapat 1992)
Aeginetia indica (French and Sherman 1 W6), Arceuthobium pusillum (Bonga 1965; Bonga 1969), Stnga hermonthica (Okonkwo l966a; Okonkwo 1966b)
Alectra sessifora (Okonkwo 1 992), Amylotheca dictyophleba (Bajaj 1 970), Arceuthobium douglasii (Blakely 1 958), Dendrophthoe falcata (Johri and Bajbj 1963; John and Bajaj 1965; Nag and John 1976; Nag and Ram 1977; R a . and Singh 199 1 ; Ram et al. 1993; Bajaj 1995;), Phoradendron tomentosum (Bajaj 1 WO), Santalum acuminatum (Barlass et ai. 1 9 8O), Snrmla philippemis (Johri and Bajaj 1 964)
Cuscuta reflexa (Baldev l959a)
untreatable disease caused by phytoplasma-like organisms (Rao and Bapat 1978). Seeds
are viable for one year, and seedlings typically have a high mortality rate.
Micropropagation could be used for large-scale production of sandalwood plants on a
commercial basis since a high fkquency of sornatic ernbryogenesis has been reported
(Bapat et al. 1990). Cell suspensions were grown in bioreactors to maxirnize embryo
production and a continuous harvest of plants fiom embryos was reported (Rugkhla and
Jones 1998; Bapat et al. 1990). Embryo formation was show to be a continuous, non-
synchronous process (Bapat and Rao 1984). Synthetic seeds were prepared by
encapsulating somatic embryos in an alginate maûix (Bapat and Rao 1988). Protoplast
fusion and gene transfer are methods that potentially can be used to enhance wood quality
and oil, dong with tolerance to salt, ârought, temperature, and disease (Bapat and Rao
1992). Protoplasts fiom hypocotyl callus and leaf mesophyll ceils regenerated into whole
plants (Bapat et al. 1985). This technique has applications for somatic hybridization and
genetic modification (Rao and Ozias-Akins 1985). Triploid plants (3n=30) were
produced following induction of embryogenesis fiom endosperm tissues (Rao and
Raghava Ram 1983). Triploids can be used in breeding prograrns and these sterile plants
are desirable where wood quality and yield are important (Rao and Raghava Rarn 1983).
Suspension cultures of sandalwood were used to study phenolic production and
formation of somatic embryos in bioreactors. Phenolics were produced d u h g the
exponential growth phase and peaked at the stationary phase (Valluri et al. 199 1).
Suspension cultures grown in a modified air-lie bioreactor produced somatic embryos Ui
large numbers (Bapat et al. 1990). These could be regenerated into plantlets, making S.
album the first tree species, according to the authors, in which complete plantlets were
obtained from suspension cultures (Lakshrni Sita et al. 198Ob). Plantlets were established
in soi1 (Rao et al. 1984) and produced normal flowers; however, plants from tissue culture
showed a high degree of somaclonal variation (variability with respect to vigor, length
and breadth of leaf, phyllotaxy and chlorophyll content) (Rao et al. 1984). The low field
survival(lO%) was attributed to exposure to ngorous field conditions at an early stage of
development (Rao et al. 1984). Efforts are underway to increase the ûequency of field
survival (Bapat and Rao 1 984).
Other members of this genus grown in tissue culture are the Western Australian
sandalwood (S. spicatum), native peach (S. acurninaturn) and the plum bush (S.
lanceolatum). Western Australian sandalwood is grown for the same reasons as hdian
sandalwood (Rugkhla and Jones 1998). Clona1 propagation of native peach and plum
bush will provide a useful adjunct to a breeding prograrn to develop edible fhits and nuts
in semiarid areas of Australia since vegetative propagation by cuttings cannot be
achieved. Somatic embryos and plantlets were recovered in S. spicatum and shoots were
produced fiom ail culhired aerial parts of seedlingsfmature tissues of native peach and
from nodal segments of plum bush (Barlass et al. 1980).
2.1.6. Scrophulariaceae
Figworts are chlorophyllous mot hemiparasites. Root parasitism bas three phases:
seed germination (radicle emergence), seedling growth (radicle and cotyledon
emergence), and haustorial contact with the host mot, which are controlled by diffeient
factors. Ln the mature seed, embryos at the cotyledonary stage have a bipolar stmcture
with both plumular and radicular poles (Okonkwo 1 992). Tissue culture studies of
members of this family were conducted to study haustoria initiation using Mwashige and
Skoog medium without plant growth regulators. The parasite determines host suitability
before forming a haustorium by secreting an enzyme that oxidatively releases a factor
60m the host root surface (the haustoria-inducing factor or HIF). The HIF, in hun, acts
as a signal for gene expression and a haustorium is initiated. Enzymatic degradation of
host cells during haustonal entry releases additional HIFs which signals M e r haustoria
developrnent when an appropriate host is found (Estabrook and Yoder 1998). HIF's were
identified and classifed into 4 groups: flavonoids (i.e. xenognosin A, xenognosin B,
formonetin), p-hydroxy acids (i.e. f e d i c acid), quinones (i .e. 2,6-dimethoxy- p-
benzoquinone, DMBQ) and c ytokinins (i .e. zeatin)( Estabrook and Y oder 1 998). The
responses of roots to HIFs were rapid and similar arnong different members of this
famil y.
Agalinis. Agalinis is a chlorophyllous, hemiparasitic root parasite found in the
southeastem USA on a number of hosts, including species of pine, poplar and oak. Fast,
exogenously regulated development of haustoria in tissue culture provided a useful
system for developrnental analysis (Steffens et al. 1986). In the majority of cases, host
root exudate was needed to initiate haustoria (Smith et al. 1993); however, there was
spontaneous appearance of some haustoria, indicating the requirement for host root
exudate was not absolute (Steffens et al. 1982). Haustoria were abundantly and rapidly
produced when seeds in vitro were exposed to host root exudate (Riopel and Musselman
1979). Two phenolics isolated fiom gum tragacanth were found to initiate haustoria.
Table 3. Responses of 23 genera of parasitic plants to tissue culture conditions.
Parasitic Plant Response in Tissue Culturea Reference
Cuscuta arvemis Shoot growth
Cusmta cmnpestris Flowers, seedlings, shoot growth
Cuscuta chinensis Shoot growth
Cuscuta japonica Flowers, haustoria, shoot growth
Cuscuta reflexa
Cuscuta myolii
LAURACEAE
Càssytha fi!#iormis
LORANTHACEAE
Amyema miquelii, A. quandang
Amyema pendulurn
Amylotheca dictyophleba
Dendrop h thoe falcata
Callus, flowers (which produced pollen and fiuit), haustoria, seedlings, shoots, somatic embryos
Callus, plantlets, seedlings, shoots, somatic embryos
Seedlings
Callus, seedlings (with haustorial discs, plumular leaves)
Callus, seedhgs (with haustorial discs, holdfasts, plumular leavcs)
Seedlings (with haustorial discs, holdfasts, plumular leavcs)
Callus, embryogenic callus, sedlings (with compound holdfasts, haus tonal discs, holdfasts, and plumular leaves), shoots, somatic embryos
Embryogenic caiius, mots, shoots, somatic embryos
Setdlings (with hausloriai discs, holdfitsts, plumuiar leaves)
Callus, seedlings (with holdfasts, plumulat laves), shoots, somatic
Binding et al. 1981
Loo 1946
Maheshwari et al. 1980
Funahashi 199 1; Funihashi et al. 1995; Tanaka et al. 1995
Baldev 1959a; Baldev 1959b; Maheshwari and Baldev 1 96 1 ; Maheshwari et al. 1980; Bhding et al. 198 1 ; Maheshwari and Sreekrishna 1982
Bakos et al. 1995
Rangan and Ranga Swamy 1969
Hall et al. 1987
Johri and Bajaj 1964; Hall et al. 1987;
Johri and Bajaj 1964; Bajaj 1970;
Johri and Bajaj 1962; Johri and Bajaj 1963; Johri and Bajaj 1965; Bajaj 1966; Bajaj 1968; Johri and Nag 1968; Bajaj 1970; Nag and Johri 1976; Nag and Ram 1977; Ram and Nag 1986; Ram and Nag 1988; Ram and Singh 1991; Ram et al. 1993; Bajaj 1995
Nag and Johri 1969; Nag and Johri 1976
Johri and Bajaj 1964
Bhojwani and John 1970
Parasitic Plant Response in Tissue Culturea Re ference
Tapinanthus bangwensis,
Taillus euneutus
T'axillus vestitus
OROBANCHACEAE
Aeginetia indicu
Chtanche iubulosa
Orobanche aegyptiaca
SANTALAC EAE
Exocarpos cupressi/ormis
Osyriis wigh tiana
Santalum acuminatum
Santaium album
Santalum lanceolatum
Santalum spicantm
SCROPHULARIACEAE
Agalinis purpurea
Alectra orobonchoides
Alectra sessljlora
Alectra vogelii
embryos
Cailus, seedlings (with holdfasts, plumular leaves), shoots
Callus, seedlings, shoots
Callus, seedlings, shoots, somatic embryos
Seed germination
Callus, shoots
Callus, seedlings, shoots
Callus, mots, shoots, somatic embryos
Roots, shoots
Callus, embryogenic cailus, micmalli (colonies h m protoplasts), plantleu, mots, seedlings, shoots, somatic embryos
Shoots
Planticts, somatic embryos
Haustoria
Seedlings
Callus, stedlings
Rwts, scedlings, shoots
Nag and Johri 1976
John and Nag 1970; Nag and Johri 1976
French and Sherman 1976
Rangan 1965; Rangan and Ranga Swamy 1968
Ranga Swamy 1963; Ben-hod et al. 199la; Ben-hod et al. 1991b
J o b and Bhojwani 1965Bhojwani 1969
Johri and Bhojwani 1965
Barlass et al. 1980
Ranga Swamy and Rao 1963; Rao 1965; Bapat and Rao 1970; Rao and Bapat 1978; Lakstimi Sita et al. 1979; Lakshmi Sita et ai. 1980a; Rao and Bapat 1980; Rao and Raghava Ram 1983; Bapat and Rao 1984; Bapat et ai. 1985; Rao and Ozias-Akins 1985; Bapat and Rao 1988; Bapat et al. 1990; Vaiiuri et al. 199 1 ; Rao and Bapat 1992; Bapat et ai. 1996; Rugkhla and Jones 1998
Barlass et ai. 1980
Rugichla and Jones 1998
Riopel and Musselman 1979; Steffens et al. 1982
Tmtmann and Visser 1987
Okonlrwo 1975; Trautmruin and Visser
Parasitic Plant Response in Tissue Culture' Reference
Melampynrm Iineare
Sopubia delphinijdia
Striga asiatica
Striga euphrasioides
Striga gesnerioides
Striga hennonthica
VISCACEAE
Arceuthobium douglasii
Arceuthobium pillum
Arceuthobium tsugeme
Phoradendron jlavescens
Phoradendron tomentosum
Viscum album
Flowers (which produced viable seeds), plants
Callus, flowen, haustoxia, roots, seed germination, seedlings, shoots
Flowers, seedlings, shoots
Caiius, flowers, baustoria, roots, seedlings, shoots
Endophytic system in host callus
Branched radicleu, callus, embryoids, seedlings (with holdfasts, haus tonal wedges)
Callus, seedling s (with holdfas ts)
Seediings
Callus, seedlings (wiih hoidfasts, plumuiar leaves), shoots
Callus, secd germination
Cantlon et al. 1963
Shivaana and Ranga Swamy 1976
Williams 196 1 ; Chidley and Drenaan 1987; Smith et al. 1990; Okonkwo 199 1; Woif and Timko 199 1;Okonkwo 1992; Wolf and Timko 1992; Cai et al. 1993; Babiker et al. 1994
Ranga S wamy and Rangan 1966
Okonkwo 1982
Okonkwo l966a; Okonkwo l966b; Okonkwo 1970
Blakcly 1958
Bonga 1965; Bonga 1968; Bonga and Chakraborty 1 968; Bonga 1969; Bonga 197 1 ; Bonga 1974
Deeks et ai. 1997
Calvin 1966
Bajaj 1970
Becker and Schwarz 197 1 a; Becker and Schwarz 197 1 b; Fukui et al. L 990
' Refer to Fig. 2
These were xenognosin A and xenognosin B, which belong to the family of compounds
having roles as antibiotics and phytoalexins (Steffens et al, 1982).
Alectra. Alecfra is a root herniparasite that attacks cowpea, groundnut, mung
bean, soybean, common beau, runner bean, chickpea, and other legumes in AWca, India,
Burma, China, and the Philippines (Parker and Riches 1993). The tiny seed contains at
one end of the endospenn a heart-shaped embryo with a plumular and a radicular pole.
Seed germination (radicle emergence) occurs only afier soaking in water for a period of
t h e at a certain temperature (conditioning), followed by stimulation through exposure to
host root exudate. Seedlings in vivo do not develop beyond radicle emergence unless the
radicle penetrates into the host root conductive system. The shoot then grows, becoming
green and les@, and produces flowers in the light. In tissue culture, gemiinated seeds
were cultured beyond radicle emergence in the absence of a host and only minera1 salts
and sugar were required for seedling growth; however, haustona were not formed,
indicating host presence was required. Plant growth regulators were not required in tissue
culture, suggesting A l e c ~ a tissues had the proper interna1 balance. If either iAA or
kinetin was added, this led to disorganized growth as levels then became supra-optimal
(Okonkwo 1975).
Melarnppm. Mdampyrum is an annual herb found in North America and
Europe that parasitizes pine, poplar, oak, aspen, and maple (Cantlon et al. 1963). Seed
germination occurs in late falVearly winter aAer a 2-month cold treatment, and once the
mot system is established, the plants remain dormant over the winter. Root growth
resumes in the spuig, with comections to the host occurring by chance (Cantlon et al,
1963). Once haustonal penetration of the root OCCUIS, contact is made with both xylem
and phloem of the host. Parasitism is not obligatory since plants can complete their life
cycle without a host. Melampyrum plants with flowers and viable seed were produced
fkom seedlings on nutrient agar without plant growth regulators. Growth was not as
Mgorous in tissue culture but this could have been due to the absence of an essential
metabolite or the confining culture tube (Cantlon et al. 1963).
Sopubia. Sopubia is a parasite of 40 species of flowering plants in India. In
nature, seed germination (radicle emergence) and seedling development (emergence of
radicle and cotyledons) requue a cold treatment, presence of moisture, host
carbohydrates, and light. Seedlings developed in culture without exogenous plant growth
regulators but sucrose was required for shoot morphogenesis. Light was essential for
germination, suggesting the phytochrome system may be involved in seed germination
and seedling development (Shivanna and Ranga Swamy 1976; Sahai and Shivanna 1985).
Striga. Striga is the most economically important member of this family of
parasitic plants, which attacks cered crops, particularly sorghum, pearl millet, rice,
maize, as well as tobacco and sugarcane in Afiica, India, Asia, Australia, and some parts
of the USA (Musselman 1980). Infection r e d t s in chlorosis, wilting, stunting, and
death, with losses ranging h m slight to 100 percent (Agrios 1997). Afier a connection is
established between host and parasite, the parasite exhibits a holoparasitic subterranean
stage of development at which time damage is inflicted. The parasite then emerges fiom
the soil, develops chlorophyllous shoots (hemiparasitic stage) and produces flowers and
seeds (Bagonneaud-Berthorne et al. 1995). If a connection is not established between host
and parasite, the seedling dies (Wolf and Timko 1992; Cai et al. 1993). In nature, seeds
germinate after several months of pst-harvest ripening followed by 6-1 0 days of water-
preconditioning, and then exposure to a germination stimulant (chexnical signal)
containeci in the root exudate of the host plant (Wolf and Timko 1991). Specific
compounds, such as 6-substituted aminopu~es, strigol, and strigol analogs (GR-7 or
GR-24), were shown to be seed germination stimulants (Worsham et al. 1959; Cai et al.
1993). In tissue culture, kinetin, gibberellin or coumarin replaced the host stimulus. Root
extract lacking a germination stimulant failed to promote seed germination (Williams
196 1; Worsham et al. 1959).
Potentially, tissue culture of Striga could be used to evaluate toxicity of herbicides
prior to field evaluation (Cai et al. 1993) or effects of nitrogen sources, nuûients and
other chernicals on seedling phy siology~biochemistry (Okonkwo 1 99 1). The herbicide
2,4-dichlorophenoxyacetic acid (2,4-D) prevented ernbryo differentiation when applied at
the time of seed germination prior to haustorium attachment (Cai et al. 1993). Resistance
mechanisms of the host can be investigated in vitro using biochemical and cytological
approaches (Lane et ai. 199 1 ).
In tissue culture, S. asiatica seedlings developed on simple, inorganic, defined
media with salts and sucrose only; plant growth regulators, vitamins, casein hydrolysate,
and meso-inositol did not irnprove growth (Okonkwo 1982; Okonkwo 199 1). A
germination stimulant (Le. exudate) was required for germination, but once the seeds had
germinated, seedlings developed without connections to the host (Chidley and Dreman
1987; Worsham et al. 1959). Genninated seeds of S. eriatica fomed haustona afta
exposure to 2,6dhethyoxy-p-benzoquinone (DMBQ), a haustoria-inducing factor that
was isolated fmm mots of the host plant Sorghum bicolor. The parasite root meristem
needed exposure to 1 0 ~ M 2,6-DMBQ for 6 hours to induce haustoria (Smith et al. 1990).
Culture of parasite roots was achieved, representing a first report for a parasitic
plant according to the authors (Wolf and Timko 199 1 ). Cultured roots could be used in
place of intact radides fiom parasite seedlings to study haustorial development suice
haustoria developed on cultured roots. There was a difference in haustorial size, with
those produced in cultured roots (in vitro) being larger than those in intact parasite
radicles (in vivo). While protein synthesis was similar in treated (2,6-DMBQ application)
root cultures versus intact radicles, there were differences between treated and untreated
root cultures (Wolf and Tirnko 1992). Three classes of proteins were reported after 2,6-
DMBQ application: those that increased in both root cultures and radicles, those that
increased in radicles only, and those that did not increase in either root cultures or radicles
(Wolf and Tirnko 1992).
An in vitro system was developed to study the mechanism of host resistance.
Host roots (Le. cowpea) were placed on glass fiber filter paper moistened with nutrient
solution and germinated S. gesnerioides seeds were placed on top. The parasite radicle
tips swelled upon contact with the host and radicular hairs were produced. After
penetration of the host stele, tubercles (swollen portion of haustorium) developed and
stems emerged fiom the tubercle apex. This non-destructive system allowed for
continuous observation and infected plant material could be easil y removed for
cytological studies. This approach was useful for screening cowpea germplasm for new
sources of resistance (Lane et al. 199 1).
The involvement of thdianuon and ethylene in seed germination of Striga was
studied (Babiker et al. 1994). Thidiazufon (a substituted urea with cytokinin-like activity
with use as a Cotton defoliant) S U ~ P ~ W ~ auxin metabolism. Auxin and cytokinins have
been postulated to stimulate the production of ethylene, leading to seed germination. As a
result, hidiazuron application enhanceci seed germination and it has potential (in
combination with auxin) for application as a germination stimulant. A novel approach
would be to screen chemicals for activity on Striga based on their ability to induce
ethylene biosynthesis (Babiker et al. 1994).
Two different types of seedlings developed depending on the medium used in
tissue culture. Parasitic-type seedlings of S. asiatica formed on host-conditioned media
while non-parasitic-type seedlings developed on control media (Cai et al. 1993). Parasitic
seedlings had a well-developed shoot system, haustoria and tubercles, while non-parasitic
seedlings had adequate root development but poor shoot development and no haustoria or
tubetcles were formed.
2.1.7. Viscaceae
Arceuthobium (dwarf rnistletoe). Arceuthobium is a destructive parasite of
commercially important forest trees which is the most evolutionarily specialized genus of
Viscaceae (Harvey 1967). The seeds have a small embryo, with the cotyledonary end
enclosed in an endosperm. Durhg germination, poorly developed cotyledons emerge that
do not become the first foliage leaves. Seeds genniaate on host tree branches and produce
radicles which grow dong the surface of the bark. Evennially, a mass of cells, the
"holdfast", is formed at the tip of most radicles, which anchors the parasite to the host and
then penetrates it. AAer penetration, a network of branching filaments calleci the
"endophytic system (Bonga a d Chakraborty 1968) develops, which is a long-lived
ramifjmg absorbing system that saps the host as long as the host is alive. Arceuthobium
is considered to be an advanced parasite, as it has a vestigial shoot apex (the shoot apex
cannot develop into a shoot), a root cap is absent, and terminal haustoria are forrned.
A. pusillum. A. pusilluna is a parasite on black and white spnice in eastem North
America (Bonga 197 1) that fomed callus, embryoids and seedlings with radicles
(branched, untiranched) and holdfasts (branched, unbranched) in culture. The holdfasts
were regularly shaped, larger, and more differentiated than those produced in vivo (Bonga
1969), and were induced by high auxin levels early during development (Bonga 1968).
Seed explants required awin (Bonga 1968; Bonga 1969) whereas embryo and seedling
explants did not (Bonga 1968; Bonga and Chakraborty 1968). Callus developeà on
Harvey's medium and had haustonal tendencies i.e. bulging cells (Bonga 197 l) , and the
endophytic system was formed in vitro as branched radicles that resembled cortical
strands were observed (Bonga 1974; Bonga and Chakraborty 1968).
Seedlings of western hernlock dwarf mistletoe (A. tsugense), with radicles and
holdfasts, developed in culture (Fig. 3) and callus developed fkom split radicles and split
holdfasts (Deeks et al. 1997).
Douglas-fir stems idected with A. douglasii have been cultured and callus was
produced that contained the mistletoe endophytic system (Blakely 1958). This species of
mistletoe has not yet been cultured in vitro without a host.
Phoradendron (true or les@ mistletoe). Phoradendron is a stem parasite of
conifm, pear, pecan, walnut, citrus, and coma in North America, South Arnerica and the
Caribbean (Parker and Riches 1993). T d infections d u c e tirnber quality and promote
fiingal and insect aîtack, which causes fiuther damage (Parker and Riches 1993).
Figure 3. Arcmthbium tsugense seed cultures on Harvey's medium. A. Elongating radicle after I month in cultun. B. Further elongation of the radicle dler 2 months in culture. C. Formation of a spherical holdfaa at the tip of the radicle after 5 months in culture. Scale bar represents 1 mm.
Phoradendron is considerd to be a primitive parasite, as the shoot apex can develop into
a shoot and there are no terminal haustoria. In tissue culture, seedling vascular anatomy
and the stages between germination and formation of an endophytic system were shidied
(Calvin 1966). Callus, shoots and seedlings were produced (Bajaj, 1970), but the
seedlings did not possess haustorial discs, indicating that the shoot apex was inactive and
the endophytic system poorly developed (Bajaj 1970).
Viscum. Vismm is a parasite with a wide host range including deciduous trees,
h i t trees, nibber, walnut, p e r s h o n , fi and pine. It occurs in Europe, Australia, Asia,
Africa and China (Parker and Riches 1993). The European mistletoe (Viscum album)
may contain anti-cancer agents (Kuttan et al. 1990), but the parasitic association and slow
growth make large-scale production difficult. Therefore, tissue culture was used to obtain
a homogeneous source of material (callus) from which extracts were obtained and found
to contain active ingredients, such as acetylcholine, histamine, flavonoids, lectins, and y-
aminobutyric acid (Becker and Schwm 197 1 a). These chernicals were used in products
to treat tumors (Kuttan et al. 1 990; Jurin et al. 1 993), arthritis, hypertension and
arteriosclerosis (Becker and Schwarz 1 97 1 a). Mistletoe callus was placed directi y on top
of beech (Fagus crenuto) callus to sîudy cell-ce11 interactions between the mistletoe and
its host in vitro, but the calli grew together without any contact inhibition (Fukui et al.
1 990).
2.2. APPLICATIONS OF TISSUE CULTURE: PRESENT AND FUTURE
The seven families of parasitic plants that have been described in this review show
similanties and differences in response to tissue culture. A range of explant types have
been used (Figure 2), with the predominant ones being seeds and ernbryos. The type of
media used dso varied (Table 2), with White's medium being the most widely used for
al1 families of parasitic plants. The results obtained in tissue culture have differed, but
seedlings have been produced from germinated seeds in al1 families. Somatic ernbryos,
however, were only produced in Santalaceae, Loranthaceae and Convolvulaceae, and
flowers only in Dendrophthoe (Loranthaceae), Cuscuta (Convolvulaceae), Striga and
Melampyrum (Scrophulariaceae). Haustoria (holdfasts in mistletoe) were produced in
Convolwlaceae, Scrophulariaceae, Loranthaceae and Viscaceae (Table 3).
Arnendments such as coconut milk, casein hydrolysate, yeast extract and
watermelon juice were used as nutrient supplernents, and it was hypothesized that these
amendments replaced the host stimulus in culture (Rangan 1965). Coconut milk was
included in experiments with al1 families except Loranthaceae and Lauraceae, whereas
casein hydrolysate was evduated for al1 families except Lauraceae. Yeast extract and
watennelon juice have had limited use- the former for Santalum, Phoradendron and
Cuscuta and the latter for Orobanche. Watermelon juice induced shoot differentiation
fiom caiius in Orobanche (Ranga Swamy 1963). In Orobanche and Cistanche, the need
for cornplex media was related to the undifferentiated statu of the embryo.
The main plant growth regdators employed in tissue culture were awins and
cytolinins, either alone or in combination. Gibberellins were included for somatic
embryo production in Suntulum (Lakshmi-Sita et al. 1979), growth of shoot tips in
Cuscuta (Maheshwari et ai. 1980), and production of callus and seedlings in Orobanche
(Ranga Swamy 1963; Ben-hod et al. 199 1). Auxin alone, cytokinin alone, and auxin with
cytokinin, initiated callus, shoots, mots, seedlings, plantlets, and somatic embryos.
Plantlets and somatic embryos developed with auxin or cytokinin alone. Kinetin was
used to induce bud differentiation in embryos of Dendrophthoe (Nag and Ram 1977) and
was the most effective growth regulator in Tapinanthus. It was hypothesized that both
kinetin and gibberellin replaced the host stimulus in culture (Williams 196 1). Plant
growth regulaton are not always requùed in tissue culture of parasitic plants and media
lacking them c m be used for seedling production in al1 families except Santalaceae and
Loranthaceae.
The objective of tissue culture experiments has varied depending on the parasitic
plant being studied. In sandalwood, the objective was large-scale production of
sandalwood plantlets through micropropagation. Preliminary trials showed that these
plantlets grew more quickly and produced scented heartwood faster, reducing time to
harvest and increasing economic retms (Lakshmi Sita 199 1). Mistletoe was cultured to
investigate anatomy and development, host-parasite interactions, germination,
polyembryony, the effects of hormones and nuûients, and production of anti-cancer
agents. Mistletoe seeds geminated and holdfasts were formed in culture, indicating that
these stages were not dependent upon contact with a living host (Bhatnagar 1987). Tissue
culture experiments with Striga and Orobanche were conducted to understand how to
conml these destructive parasites by ùivestigating signals that occurred between the
parasite and its host.
Host-parasite interactions can be studied at the cellular or molecular level. There
are two examples where host and parasite were grown together in tissue culture. Callus
of Viscum album was placed on beech callus to investigate the interaction in vitro but no
contact inhibition occurred (Fukui et al. 1990). Tomato roots were aseptically infected
with Orobanche callus (Ben-hod et al. 199 1 b) or seedlings (Losner-Goshen et al. 1996)
and cdlus behaved like a radicle and formed a haustoriurn upon contact. This system
may simpliQ the study of chernical signals between host and parasite, including those for
haustorial initiation and host attachent.
Cwrent tissue culture research on the more destructive parasitic plants is focused
on characterizing signals and induction of gene expression at the molecular level.
Emphasis is on determining signals for parasite development, including seed germination
and haustorium formation. Haustoria-induchg factors, such as flavonoids, p-hydroxy
acids, quinones and cytokinins, were show to induce haustoria formation (Estabrook and
Yoder 1998). In Striga, a low molecular weight quinone (2,6-DMBQ) was shown to act
as a signal for haustorium formation (Smith et al. 1990). If the signals for seed
germination are found, induction of germination in the absence of the host or complete
prevention could be achieved (Press et al. 1990). Germination in the absence of a suitable
host root is effectively suicidai, and chernicals that trigger germination have potential as a
means of control (Parker and Riches 1993). If the signals for haustorium initiation are
found, they could be used to inhibit production of haustoria by preventing gene
expression.
In the development of control procedures, chernicals and herbicides can be
screened in tissue culture prior to field evaluation (Cai et al. 1993). For example, the
application of 2,4-D prior to haustorium formation in Striga may prevent embryo
differentiation (Cai et ai. 1993). Prevention of embryo differentiation by synthetic auxins
and possibly other herbicides is an approach that merits M e r investigation.
Potential biological control agents of parasitic plants, such as fungi or insects, CM
also be screened using the in vitro systern. Parasite plant explants could include callus,
embryos, seedlings, plantlets, isolated organs, or parts of organs (Diner and Karnosky
1987). Cment research in our laboratory is focused on producing callus of dwarf
rnistletoe, Arceuthobium tsugense, which can then be challenged with fwigi as part of an
in vitro screening for potential biocontrol agents (Deeks et al. 1 997; Shamoun 1 997).
Another potential control method for dwarf mistletoe is genetic resistance. Scharpf
(1987) found that heritable resistance in Jefney pine infected by dwarf mistletoe was
characterized by a reduction in number of infected trees, infections per tree, and infection
intensity. Smith et al. (1 993) describecl evidence for resistance mechanisms operating
within the host branches of western hemlock infected with dwarf rnistletoe. Resistance
appeared to result from the reduced ability of the penetrating structure to enter the host
cortex or early demise of the structure before infection, both of which could be studied in
tissue culture.
Progress in the area of tissue culture of parasitic plants provides additional
researcb opportunities. Studies on the growth, physiology and host-parasite relatiomhip
of mistletoe would be valuable, in addition to studying shoot bud development and anti-
cancer agent accumulation. In ûrobanche, the role of enzymes and the nature of
chernical sipals between host and parasite need to be determined. The role of pectin
methylesterase during attachment of the parasite to its host warmts M e r work. Mutant
plants lacking pectin methylesterase or in which the gene was disrupted could be used to
study pathogenesis. Polygalacturonase, yet to be characterized, could be another enzyme
similar to pectin methylesterase that is involved in degradation of ce11 wall pectins which
bind adjacent host cells (Ben-hod et al. 1 997). Since Santalum can be regenerated fkom
protoplasts, friture work could utilize somatic hybridization via protoplast fusion and
genetic modification via gene ûansfer. Development of control methods for destructive
parasitic plants such as Shiga and Arceuthobium would be valuable. In Striga, chemicals
could be screened for their ability to induce ethylene biosynthesis and affect seed
gexmination. In Arceufhobium, biocontrol fungi and genetic resistance are potentially
valuable contrd methods that require additional research. The search for signals
promoting seed germination and houstorium fonnation in other destructive parasitic
plants may prove useful. The identification of chemicals that inhibit embryo
differentiation or induce suicida1 germination (Bagomeaud-Berthorne et al. 1995) may
also yield promising results.
From a tissue culture perspective, parasitic plants are an intriguing group of
organisms in which to study the developmental events during various stages of
differentiation, growth and development. The use of this information is wide-reaching;
methods to control destructive parasitic plants can be developed, plants with economic
value can be propagated, and plants with therapeutic value such as Viscum album may
yield important drugs for cancer therapy. Finally, the availability of tissue culture
procedures for many of the parasitic plants couid stimulate studies on foreign gene
insertion or effects of gene disruption through antisense technology on parasite behaviour
or survivai. These powerful molecular techniques should shed light on some of the
intriguing host-parasite relationships described here, with the ultimate goal of controlling
these destructive parasites.
Note: Chapter 2 has been published in: In Vitro Cell. Dev. Bio1.-Plant (1999) 35:369-38 1 .
CHAPTER 3
LN VITRO GERMINATION AND DEVELOPMENT OF WESTERN
HEMLOCK DWARF MISTLETOE (ARCEUTHOBIUM TSUGENSE
SUBSP. TSUGENSE)
3.0. INTRODUCTION
Mistletoes (members of the family Viscaceae and Loranthaceae) are widespread
and destructive parasitic flo wering plants. Dwarf mistletoes (A rceuthobium spp.
(Viscaceae) parasitize conifers of the Pinaceae and Cupressaceae and cause reductions in
vigour, growth, seed production and wood quality as well as tree mortality (Hawksworth
& Wiens 1996). Control of western hemlock dwarf mistletoe has been hindered by poor
knowledge of the biology of the parasite and the host-parasite relationship; current control
methods include silvicultural, chernical, genetic, and more recently, biological conuol
with h g i (Shamoun 1997). A new management tool is necessary as the change in
forestry practice has lead to a reduction in cut block size and a move to partial cutiing
operations. These operations increase the ratio of edge trees to seedlings, thus enhancing
mistletoe spread and damage (Shamoun 1997).
There are two subspecies of hemlock dwarf mistletoe (A. tsugense (Rosend.) G.N.
Jones): western hemlock dwarfmistletoe (A. mgense subsp. mgense) found on westem
hemlock (Tsuga heterophylla (Raf.) Sarg.), and mountain hemlock dwarf mistietoe (A.
tsugense mbsp. mertensianae) found on mountain hemlock (Tsugo mertemiana (Bong.)
Cm.). Two races of western hemlock dwarfmistletoe have been described (Hawksworth
& Wiens 1996). The western hemlock race attacks western hemlock and the shore pine
race attacks shore pine (Pinus conforta Dougl. ex Loud.var. contorta). A. tsugense subsp.
tsugense is found near the North Amencan West Coast of Canada (primarily in British
Columbia) and the United States (Alaska, Washington, Oregon, and
Cali fomia)(Hawksworth & Wiens 1 996).
Seeds of dwarfrnistletoe are atypical. There are no tnie ovules in either the
Viscaceae or Loranthaceae and consequently, there is no testa and therefore no tnie
"seed". The "seed" is an embryo embedded in a chlorophyllous endosperm surromdeci
by a layer of viscin (mucilage)(Hawksworth & Wiens 1996). The embryo itself is
chlorophyllous, with a meristematic radicular apex. The cotyledons of the embryo are
vestigiai and there is no root cap (Hawksworth & Wiens 1996). The seed itself is
photosynthetic (Muir 1975) and the growing radicle has stomata. This autotrophic
capability increases seed longevity beyond the availability of stored nutrients
(Hawksworth & Wiens 1996).
Donnancy in the traditional sense does not exist in dwarf mistletoes (Hawksworth
& Wiens 1996). In temperate regions, growth of the radicle ceases during periods of
unfavorable conditions, but the radicular apex r e m e s growth when favorable conditions
retum, which include warmer temperatures (1 SOC to 20°C)(Gill & Hawksworth 196 1 ;
Scharpf 1 WO), presence of light (Scharpf 1970; Wicker 1974), and an unintermpted
period of high humidity (Bonga 1969). Likewise, traditional "afler-ripening" (expomre
to low tmperatures) is not needed by dwarf mistletoe seeds (Scherpf 1970
Germination begins with the initiation of meristematic activity at the radicular
apex. The radicle grows until it reaches an obstruction, at which t h e a holdfast forms.
The holdfast is a smooth sweiling at the distd end of the radicle (Baranyay et ai. 197 1).
In the center of the holdfast, a region of intense menstematic activity develops to form a
"penetration wedge", which enters the host cortex by exerting mechanical pressure
(Scharpf & Panneter 1967), and develops into a rootlike endophytic system which then
ramifies throughout the host bark.
Several members of the Loranthaceae have been cultured in vitro, and production
of shoots, roots, callus, seedlings, and somatic ernbryos has been reported (John & Bajaj
1964; Hall et al. 1987; Nag & Johri 1976; Onofeghani 1972). Only four members of the
Viscaceae, including A. pusillum, have been cultured in vitro, and callus, shoots,
seedlings, and embryoids were produced (Bajaj 1970; Becker & Schwm, 197 1 a, 197 1 b;
Bonga 1965,1968, 1969, 197 1, 1974; Bonga & Chakraborty 1968; Calvin 1966; Fukui et
al 1990). Since western hernlock dwarf mistletoe (A. tsugense subsp. tsugense) has n ~ t
previously been grown in tissue culture, the objectives of this study were to 1) investigate
whether growth in vitro was possible, 2) determine the optimal conditions for growth,
germination, and production of the early seedling stage, and 3) produce callus
(undifferentiated tissue). The long-tm goal of this research is to produce cailus and
geminated seeds for in v i h ~ screening of potential h g a l biological control agents that
could inhibit dwarf mistletoe development in nature.
3.1. MATERIALS AND METHODS
3.1.1. Seed collection
Western hemlock branches containing clusters of ripe dwarfmistletoe h i t (A.
tsugense subsp. tsugense) were collecteci in early October fiom Texada Island, B.C.
(4g045' N, 124'30' W), placed in plastic bags, and stored at 4OC pnor to seed extraction.
The fruit were squeezed between the thumb and forehger to expel the seeds, which were
collected onto a large piece of cotton cloth. Seeds were then transfmed to petri dishes
and left to air dry for 48 hours before storage at 4°C.
3.1.2. Pre-screenhg seeds for viabiiity
Prelimininary investigations were done to examine why some seeds tumed yellow
when placed on tissue culture media. The destructive 2,3-5 -tnphenyl tetnizolium chloride
test, modified from Wicker ( 1974), was used: the endocarp was removed from a sarnple
of green and yellow seeds and seeds were placed in a via1 containing a 1 % buffered
tetrazolium solution (5 gram of tetrazolium salt, 1.8 16 gram of KH2P04 and 2.408
gram of Na2HPO4 2H20 in 500 ml of distilled water) and incubated in the dark for 24
hours at room temperature. nie endospem and embryo were checked for the presence of
a red precipitate, indicating viability. The result was that green seeds were viable and
yellow seeds were not. Mer 3 months of storage, seeds were pre-screened for viability:
seeds were surface-sterilized by sbaking for 30 minutes on an orbital shaker (VWR
Scientific) at 100 RPM in 3% hydrogen peroxide. In a laminar flow hood (SterilGARD@
II by The Baker Company, Sdord, Maine, USA), seeds were rinsed three times in a
sieve with distilled deionized water and then transferred to petri plates containing
moistened Whatman # 1 filter papei- and incubated at 4OC with a 12-hour photoperiod
(cool white fluorescent, 138 pm~lm~~s ' ' ) until a color change was obsened (about 4
days). Green seeds were left to air-dry in the laminar flow hood (about 2 hours) and then
sealed within pehi plates and stored in the dark at 4°C until needed for tissue culture
experiments. The average seed viability of dwarf mistletoe seeds collected fkom Texada
Island was 35%.
3.1.3. Media
Two different tissue culture media were used: Harvey's medium (HM)(Harvey,
1967) and modified White's medium (WM)(Ranga Swarny 1961) containing two
additional supplements: 15% coconut milk (vh) and 0.5 g 1" casein hydrolysate which
had been s h o w to improve callus growth (Deeks et al. 1998). Prelhinary experiments
using several different media, including DCR (Gupta Br Durzan, 1985)' Harvey's (Harvey
1967)' Liway's (Litvay et al. 1985)' Murashige and Skoog (Murashige & Skoog 1962),
and modified White's with and without supplements (Ranga Swamy 196Q had shown
that Harvey's medium and modified White's medium were superior for growth and callus
production (Deeks et al. 1998).
3.1.4. Experimentaî design
Viable seeds w m aseptically trmsferred to the tissue culture media. Regular
transfers were not made as these were found to inhibit growth (data not shown). Cultures
were maintained at 15°C (HM) or 2OT (HM and WM) under both light and dark
conditions in a five-way factorial experiment (Zar 1996) testing the effects of media,
temperature, light, 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine
(BAP). Levels of 2,4-D included O pM (O mg 1-'), 4.52 x lo4 FM (0.1 mg 1-'),2.26 x 10-~
p M (0.5 mg 1.') and 4.52 x IO-' pM (1 mg rL). Levels of BAP included O pM(1 mg 1-'),
4.44 x 1 Cl4 pM (0.00 1 mg FI), 4.44 x 1 O" pM (O. 1 mg 1") and 4.44 x 10" (1 mg 1-l).
There were 3 0 seeds per treatment for HM (6 replicates with 5 seeds per plate) and 10
seeds per treatment for WM (2 replicates with 5 seeds per plate). A 12- hour photoperiod
at 5 pn01m'~s" intensity (cool white fluorescent) was used for the light conditions.
3.1.5. Light microscopy
During and afier the tissue culture experiment, selected specimens were fixed in
2.5% glutaraldehyde in 0.075M phosphate buffer, pH 7.2. The specimens were
dehydrated in alcohol, embedded in Technovit 7 1 00 (2-hydroxyethy lmethacrylate),
sectioned using a pyramitome ( 2 ~ sections), stained in 0.05% aqueous toluidine blue
for 1 min and anatomy was examined under the light microscope. The protocol used was
provided in the Technovit 7100 kit fiom Kulzer, distributed by Marivac Ltd. Halifax,
Nova Scotia, Canada.
3.1.6. Statisticd analysis
Data was analyzed using Statistical Analysis System (SAS)(SAS htitute Inc.,
Cary, NC 1985). Quantitative data were analyzed with the General Linear Models
procedure (Zar 1996) to test for effects of media, temperature, light, 2 ,eD and BAP on
radicle length (1 -4 months), holdfast diameter ( 1-4 months) and callus diameter ( 1 -7
months). Diameters of holdfasts and calli were calculated by adding the length
measurement to the width measurernent and dividing by 2 (Taylor and Secor, 1992). The
Bonferroni t-test was u s d for multiple cornparison testing. Qualitative data were
analyzed with the Frequency procedure to give the fiequency of the trait (radicle color,
radicle direction, holdfast shape, holdfast color, and callus color) according to the specific
treatment for the duration of the experhent (7 months).
3.2. RESULTS AND DISCUSSION
32.1. Radiele development
Mistletoe seeds germinated within the first week aller plating and the slow-
growing radicles reached a length of 1- 12 mm afier 4 montiis in culture on both types of
media and al1 plant growth regulator combinations, and continued to elongate beyond 7
months (Fig. 4a). Germination in nature requires the presence of water (Bonga 1 W2),
which is readil y available in tissue culture media (Bonga 1 96 8). A. tsugense genninated
more quickly (1 week) compared to A. pusillum (3-6 weeks)(Bonga 197 1). Perhaps the
initial pre-screening for viability under moist conditions with a 12 hour photoperiod
mimicked spring conditions and promoted rapid seed germination. In addition, the
hydrogen peroxide used to sterilize dwarf mistletoe seeds could have acted as a
germination stimdant (Wicker 1974). Hydrogen peroxide also increased germination in
western white pine seeds (Pite1 and Wang, 1985). The low viability (35%) of dwarf
mistletoe seeds may be due to immaturity and poor embryo development; prescreening
for viability is therefore recommended prior to initiahg tissue culture experiments.
Radicle length was significaatly affected (P=0.001) by medium, temperature,
light, 2,4-D and BAP during the first 4 months. Significantly (P=0.02) longer radicles
were produced on White's medium compared to Harvey's medium. Temperature also
had a significant (P=0.02) effect, with 2WC being better than 15OC for production of the
longest radicles. Light increased radicle length in both media, and with al1 combinations
of plant growth regulaton in months 2 and 3 (P=0.002). Both 2,4-D and BAP reduced
radicle growth. In plates without 2,4-D, radicles were significantly (W0.05) longer than
in plates with O. 1,O.S or 1 mg 1-' 2,4-D. Radicle lengths on plates with 0.1 mg 1" 2,4-D
were significantly (P<0.05) longer than those on plates with 0.5 or 1 mg 1" 2,4-D.
Radicle length was reduced with BAP, with longest radicles produced without BAP, and
radicle lengths decreased with increasing levels of BAP. This is consistent with the
function of cytokinin as a root inhibitor (Taiz & Zeiger 199 1).
Radicles were green, r d , orange or yellow in color, or a mixture of these. Green
(44%), red (35%) and orange (1 3%) were the most common, with yellow (4%) in the
lowest frwluency. The multitude of colors reflect presence of chlorophyll and carotenoid
production, found in seed plants and al1 photosynthetic organisms (Taiz & Zeiger, 199 1).
Light had an influence on radicle color, and green radicles were produced significantly
more in the dark (Pc0.00 1) for ail treatments. Red radicles were produced significantly
more in the light (P<0.001) for al1 treatments and this result was similar to findings by
Scharpf (1 970).
AAer emerging h m the seed during germination, the radicles either grew
horizontally dong the d a c e of the medium (9 1 % of the seeds), vertically in an upward
direction (3%), or down into the agar (4%). About 5% of the radicles formed tight or
loose curls, which curled over or around the seed (Fig. 4b). Radicles were mainly
unbranched; however, there was one branched radicle produced on HM with 0.1 mg 1-'
BAP in the dark at 20°C (Fig. 4c). The branch emerged fiom the side of the radicle.
The epidennis of some radicles split in culture on HM (frequency of 3%) and WM
(fiequency of 1 1%), either horizontally (i.e. perpendicular to the longitudinal axis of the
holdfast) at the tip or at the base. The majority of radicles split at the tip (Fig 4d)(58% of
radicles with splits), followed by splits at the base (1% of radicles with splits). Split
radicles appear to be unique to A. Lnigense as they were not seen in A. pusillum.
3.2.2. Boldfast development
In nature, holdfast formation is determinecl by the contours of the host bark and by
obstructions encountered by the growing mdicle (Bonga 1969). In culture, such
obstructions do not exist and the development of holdfasts does not depend on pressure
provided by obstacles, but rather on the chernical nature of the culture medium (Bonga
1969). In this study, holdfasts (smooth swellings) developed after 2 months in culture in
6.20% of the seeds on medium containhg growth regulators. Holdfasts were initiated
earlier in A. tsugense (2 months) compared to 4 months in a study of A. pusillum (4
months). This may have been due to the initial pre-screening of seeds for viability, which
may have hastened development, since holdfasts were initiated at 4 months in cases
where seeds were not pre-screened for viability (data not shown). Half of the holdfasts
developed at the tips of radicles and the other half developed by swelling of the whole
radide. Culture media influenced holdfast production, with significantly (R0.00 1) more
Figure 4. Steps in germination of mistletoe seeds and production of radicles, holdfasts and callus on HM (a-i) and WM ÿ). a. Genninated seed with elongating radicle (mow). b. Curled radicle. c. Branched radicle. d. Split radicle with callus (mow). c. Spherical holdfast ( m w ) produced at the tip of the radicle. f. Oblong holdfast ( m w ) . g. Tnangular holdfast ( m w ) . h. Holdfast split in the middle (arrow). i Split holdfast with callus ( m w ) . j. Endospenn dedifferentiation to yield callus fiom endosperm (arrow). Scale bar represents 1 mm.
of the seeds producing holdfasts on HM afler 3 months (20%) than on WM (6%)(Fig. 5).
Significantly more holdfasts (PcO.00 1) were produced in the light cornpared to the dark
and at 20°C cornpared to 15°C. Holdfast production was dso enhanced by increasing the
level of 2,4-D and BAP (Fig. 6). This suggests that holdfast formation is induced by high
levels of endogenous a w i n (Bonga 1968), since auxins can promote ce11 elongation, ce11
division, and tissue swelling (Taiz & Zeiger 1 99 1 ).
Holdfasts grew progressively larger in culture over time, with the average size
being 1.07 mm on HM and 0.95 mm on WM a h 4 months at 20°C in the light. Larger
holdfasts were produced on HM (P=O.O 17), under light conditions (P=0.04) at a
temperature of 20°C (P=0.0097) than in the dark and at 1 5°C. In general, holdfast
diameter increased with increasing levels of 2,4-D, reaching a maximum size with 0.5 mg
1-' 2,4-D. The effective levei of BAP was different, and maximum size was obtained with
0.1 mg 1" BAP. Holdfasts produced in vitro are larger, more regularly shaped, and more
differentiated than holdfasts in vivo (Bonga 1969).
Holdfasts were green, red, orange, or yellow. Production of red holdfasts was
significantly higher in the light than in the dark for al1 treatments (P<O.OO 1). The
majority of holdfasts (50-75%) were spherical (Fig. 44, about 25% were oblong (Fig. 40,
roughly 8% were aiangular (Fig. 4g), and only about 0.2% of the holdfasts were square in
shape.
When spherical holdfasts of A. tsugenre were sectioned, a compact cellular
structure was observed, with closely packed parenchyma cells surrounded by a single
epidermal layer (Fig. 7). This is in contrait to spherical holdfasts of A. pusillum, in which
Months in culture
Figure 5. Holdfast production on Harvey's medium and White's medium at 20°C. Holdfast production decreases as holdfasts split to fom callus.
Figure 6. influence of plant growth regulaton on holdfast production. Levels 1-4 for 2,4-D represent O, O.l,O.S and 1 mg r', respectively. Levels 1-4 for BAP represent O, 0.00 1 , O . 1 and 1 mg 1'' respectively. The graph represents the overall average for al1 seeds.
Figure 7. Light micrograph of a spherical holdfast produced from a swollen radicle on Harvey's medium after 7 months in culture at 2CPC light (1 mg f ' 2,4-D and I mg lm' BAP). a. Longitudinal section through a seed showing the spherical holdfast. (ec, endocarp; en, endospenn; ho, holdfast; vc, viscin cells). Scale bar represents 1 mm. b. Spherical holdfast magnified to show the closely packed parenctiyma cells surrounded by a single layer of epidemal cells. Scale bar represents O. 1 mm.
a radiating cellular structure was observed, with parenchyma cells radiating f?om the
center of the holdfast (Bonga & Chakraborty 1968). Thus, two unique features of A.
tsugense are 1 ) radicles swelling to become spherical holdfasts and 2) spherical holdfasts
with a compact cellular structure.
Approximately 14% of the holdfasts split in culture (Fig. 4i) resulting in the
development of callus around the exposed tissue. Split holdfasts with callus were also
produced in A. pusilium (Bonga, 197 1). The majority (53%) of holdfasts split in the
middle (Fig. 4h), followed by those that split at the tip (28% ) and at the base (6%).
3.23. Caiius development
Callus startecl to develop atter about 2 months in culture from radicles (Fig. 4d)
and split holdfasts (Fig. 4i) and continued to be produced for the duration of the
experiment. The percentage of seeds that produced callus reached a maximum at 7
months for al1 treatrnents (Fig. 8). Callus production was influenced by media,
temperature, light, and plant growth regulators. Cailus production was higher on WM
(38%) compared to HM (12%) over months 2-7 inclusive (P=0.041). A broad range of
media have been used for callus production in parasitic plants (see review by Deeks et al.
1999) but White's medium either in its original form (White 1943) or in modified forms
are the most commonly used. Sucrose levels in White's medium ranged nom 2% to 5%,
and supplements such as coconut milk and casein hydrolysate have been added to induce
callus fornation. Coconut milk is a natural source of organic nitrogen, has cytokinin
activity and provides a variety of complex chernicals with growtWorganogenic
stimuiating capacity (Bonga & von Aderkas 1992). Coconut milk has been used at levels
of 5-1 5%. Casein hydrolysate is a n a t d source of organic nitrogen and amino acids
- - O * - HM, 200C + MM, 20%
Months in culture
Figure 8. Callus production for al1 treatments based on percentage of seeds that produced cdlus fiom split radicles or holdfasts.
(Bonga & von Aderkas 1992) and has been used at levels of 0.4 g 1-l to 2 g 1-l, the most
common being 0.5 g 1-l.
A temperature of 20°C was significantly (P=0.014) better for callus production
than 15T, while significantly more seeds produced callus in the dark than in the light on
HM (P<0.001) but not on WM over the duration of the experiment. Growth regulators
were required for callus production and callus production was highest with 2,4-D at 0.5
mg r1 and BAP at 1 mg l?
Callus growth rate was very slow, with an average callus diarneter of only 2.23
mm on HM and 2.9 1 mm on WM afier 7 months. Larger calli were produced on WM at
20°C in the dark than in the light. The size of callus was not affected by differing levels
of plant growth regulators, as the size differences were too small to show significant
differences using statistical analysis. Fresh weight and dry weight are the two measures
used most often to monitor growth of callus cultures (Yeoman & Macleod 1977). In this
study, these measures were not used to evaluate callus growth, as they are destructive.
Callus diameter was used as a measure of growth as it is a panuneter which correlates
well with Eresh weight and dry weight (Taylor & Secor 1992).
AAer 4 months in culture on WM at 200C in the dark, there were a few seeds in
which the endosperm dedifferentiated and callus arose fiom endosperm tissue (Fig. 4j).
Light microscopy was done on split radicles that were forming cdlus. The cells of
îhe radicle were observed to be arranged in an ordaly manner and there was vascular
tissue in the central core (Fig. 9a and 9b). In the area where the radicle tip split to yield
callus, the cells were spherical and divided in al1 directions (Fig. 9b). Light microscopy
revealed the calfus to be a mass of fauly homogenous, loosely packed, unorganized
Figure 9. Light micrographs of a split radicle with callus. a. Longitudinal section of a seed with a split radicle and callus afier 4 months. The epidermis has been split into ragged patches and an unorganized &lus resulted. (ca, callus; ec, endocarp; en, endosperm; vc, viscin cells). Scale bar represents 1 mm. b. Callus cells in the area of the split. Vascular tissue is present in the center of the sample (ca, callus; vt, vascular tissue). Scale bar represents 0.1 mm. c. Enlarged region of callus showing spherical, densely stained callus cells. Scale bar represents 0.01 mm.
parenchyma cells which, when sectioned, were spherical and densely stained (Fig. 9c).
The protodenn and epidermis rolled back and were transformed into callus. Lack of
pressure (force exerted by cells on their neighbours) could have played a role in callus
formation. Pressure has an organizing effect in growing tissues while lack of pressure
results in unorganized callus growth (Brown & Sax 1 962). As the radicles/holdfasts grew
larger, the rate of growth of the intemal tissues slowed down and the pressure on the
protoderm and epideds was reduced. This presurnably initiated dedifferentiation and
callusing at the point of least pressure i.e. near the apex of the radicle or holdfast (Bonga
197 1).
3.3. CONCLUSIONS
This is the k t report of in vitro culture of western hemlock dwarfmistletoe. A.
tsugense developed differently in culture than A. pusillum. In A. mgense, no branched
holdfasts or haustonal wedges were formed, and the callus produced was not
embryogenic after 7 months in culture. In A. tsugense, spherical holdfasts had a compact
cellular structure while in A. pus i lh , a radiating cellular structure was evident. Split
radicles with callus seem to be unique to western hemlock dwarf mistletoe along with the
swelling of radicles to form sphmical holdfasts.
The successfui in vitro culture of western hemlock dwarfmistletoe may provide a
means to screen naturally occurring fungi such as NectBa neomacrospora C. Booth &
Samuels and Colletotn*chum gloeosporioides (Penz.) Penz. & Sacc. as biocontrol agents
against dwarf mistletoe (Deeh et al. 1997; Shamoun 1997). Cultures of A. mgense may
also be used in studies of host-parasite interactions in vitro and could be a usefial system
for studying genetic resistance ( W b & Rypacek 1983; Nyange et al. 1995; Ostry &
Skilling 1992) and the physiological and biochemical mechanism of the host-parasite
interactions (Cai et al ai. 1993; Press et al. 1990).
A HISTOPATHOLOGICAL STUDY OF INFECTION OF
GERMINATED SEEDS AND CALLUS OF WESTERN HEMLOCK
DWARF MISTLETOE BY CYLINDROCARPON CYLINDROIDES
AND COLLETOTRICHUM GLOEOSPOMOIDES IN DUAL
CULTURE
4.0. INTRODUCTION
Western hemlock dwarf mistletoe (Arceuthobium tsugense subsp. mgense) is a
parasite that reduces growth, vipur, wood quality and seed production of its host,
western hemlock (Tsuga heterophylla (Raf.) Sarg.). Studies of the interactions between
dwarf mistletoe and its host are difficult to conduct, since the parasite cannot be grown in
the absence of its tree host under natural conditions. However, tissue culture methods
offer promiss as a means io research the potential of alternative control methods (Deeks
et al. 1999). Several management strategies of the parasite are being researched including
silvicultural, chemicai, genetic, and biological control approaches (Hawksworth and
Wiens 1 996; Shamoun 1997; Shamoun 1999). Biological control of dwarf mistletoe with
fimgal pathogens is one promising area of research (Shamoun 1997). Several h g i ,
including Nectria neomacrospora C. Booth & Samuels (anamorph Cylindrocaipon
cylindroides Wollenw.) and Colletohichum gloeosporioides (Penz.) Penz. & Sacc. have
been found infecting swellings, shoots and berries of dwarf mistletoes in nature (Parmeter
et al. 1959; Scharpf 1964; Muir 1967; Wicker and Shaw 1968; Byler and Cobb 1972;
Funk and Bataayay 1973; Funk et al. 1973; Hawksworth et al. 1977; Muir 1977;
Hawksworth and Wiens 1996; Shamoun 1999). Studies of the interactions of these fun@
with mistletoe could be enhanced if a tissue culture system was available that was
suitable for infection and pathogenesis. Such dual cultures have been developed in other
plant-fimgal interactions (Hrib and Rypacek 198 1 ; Woodward and Pearce 1988; Sieber et
al. 1990) but not for a tree parasite such as dwarfmistletoe. The dual system must allow
growth of the parasite as well as provide a siitable substrate for fimgal growth and
CO lonization.
The objectives of this study were to develop a medium on which dual culture of
mistletoe with fungal pathogens could be conducteci and to determine the process of
pathogenesis using histopathological methods. The ultimate goal was to develop a rapid
screening assay with which to test potential biocontrol fungi against dwarf mistletoe.
4.1. MATERIALS AND MEmODS
4.1.1. Mistietoe tissue
Seeds of dwarf mistletoe were collected from Texada Island, British Columbia
(B.C.) (4g045' N, 1 24"301 W), and germinated as desdescnbed previously (Deeks et al. 1997).
Harvey's medium (Harvey 1967) containing 2,4-dichlorophenoxyacetic acid (2,4-D) and
6-benzylaminopurine (BAP) was used in experiments to induce callus formation (Deeks
et al. 1997). The cultures were maintaineci in the dark at 20°C for up to 7 months.
4.1.2. Fungrl growth
Isolations were made fiom visibly infected shoots, berries and sweilings of dwarf
mistletoe collected fkom Duncan, B.C. in 1996. AAer surface sîerilization in 95% ethanol
(1 min) followed by 10% bleach (1 min) and 3 riases (1 min each) in sterile distilled
water, infected tissues were plated onto malt extract agar (MEA, Difco Laboratones,
Detroit MI) and incubatecl at 20°C for one week. Resulting fungai colonies were
transferred to MEA slants and stored at 4OC. From this mycobiota collection, two h g i :
Cylindrocarpon cylindroides(hereafter refened to as Cylindrocarpon), and
CoZZetotrichum gloeosporioides (hereafier referred to as Co lletoh.ichum), were selected
for M e r experiment~. The selection of these two candidate fungi was based on their
performance as pro&ing biological control agents under field conditions (Shamoun
1999). Growth on several media was evaluated. The media included potato dextrose
agar (PDA, Difco ~~boratorks, Detroit MI), MEA, water agar (WA), Harvey's medium
with or without 2,4-D and BAP, and modified White's medium (WM, Ranga Swamy
196 1) with and without 2,4-D and BAP. The various media were each inoculated with a
6 mm diameter rnycelial plug of the fùngi and incubated at 20°C in the dark for 1 or 2
weeks, at which time colony diameter was measured f?om 3 replicate plates.
4.1.3. Dual culture
Harvey's medium was porned into 100 x 15 mm petri dishes, and after it had
solidified, half of the agar was cut out and replaced by 1% WA. Mistietoe seeds without
the seed coat were placeci on HM without plant growth regulators, and WA received a 6
mm diameter plug taken nom the edge of 3 week old fungal colonies on MEA. The two
plugs were placed 45 mm apart (mycelial plug in center of water agar) and fungal growth
was m m e d e w 2 days h m thre replicate plates. A f h contact of the seed was
made by b g d myceliurn, seeds were removed 1,2,3,7, and 14 days pst-infection and
prepared for light microscopy. Dual cultures were also studied using mistletoe callus and
fhgi both growing on tissue culture media. For Cylindrocarpon, HM was used and for
Colleiorrichum, WM was used, as fûngal growth was moderate on these media. Callus
was induced on mistletoe seeds as described previously (Deeks et al. 1997; 1998). A
mycelial plug was placed in the center of the plate, 30 mm away fiom the 7 month old
callus (approxirnate diameter 3.5 mm). Fungal and callus growth were measured every 2
days and samples for light microscopy were taken 0.5, 1,2,3 and 7 days post-contact. in
one expenment, the fungi were inoculated 2 weeks afier the callus was transferred. For
light microscopy, infected mistletoe seeds or callus were fixed in 2.5% glutaraldehyde in
0.075M phosphate buffer, pH 7.2, dehydrated through an alcohol series, and embedded in
Technovit 7 100 (2-hydroxyethylmethacrylate) according to the protocol supplied in the
Technovit 7 100 kit (Kulzer, distributed by Marivac Ltd., Halifa~, Nova Scotia, Canada).
Specimens were sectioned using a pyramitome (2 pm sections), stained according to the
procedure of Gutmann (1995), and examined under the light microscope. To assess the
extent of fimgal colonization of mistletoe tissues, the seeds were divided into endosperm
and radicle (Fig. IO), and the callus was divided into halves. The rating scale (O - 5 ) was
used, where O = no hyphae present in the area, 1 = < 1 % of area contains hyphae, 2 = 1-
25% of area contains hyphae, 3 = 26-50% of area contains hyphae, 4 = 5 1-75% of area
contains hyphae, 5 = 76400% of area contaios hyphae. To confirm and quantifi the
colonization data, a higher magnification was used (200 X) and the achial nurnber of
hyphae in 1 mm2 of tissue was enumerated.
Figure 1 0. Schemat ic division of germinated seed of dwarf mist letoe showing areas used in ratings of colonization by the two fbngi.
4.2. RESULTS
4.2.1. Fungal growth
Media significantly influenced colony diameter of Cylindrocarpon; best growth
was on WM followed by HM and PDA; least growth was on WA and MEA (Fig. 1 IA).
Best growth of Colletotrichum was on MEA, followed by WM and PDA; least growth
was on WA and HM (Fig 1 1 B). Plant growth regulators did not affect growth of either
fungus.
4.2.2. Duai cdture
Both fun@ had grown over rnistietoe seeds within 10- 14 days. There was no
inhibition/stimulation of fungal growth towards or away from the seeds and fungal or
seed growth in dual culture was sirnilar to controls.
Light microscopie examinations of seeds reveaied that the endospenn was
colonized faster than the radicle by both Cylindrocarpon and Colletotrichum. The areas
of the endospemi nearest to the radicle were the first to be colonized, but the number of
hyphae observed was similas in all areas examined. Degradation of ce11 walls in the
endosperm was evident as early as 2 &ys for Cyiindrocarpon (Fig. 12ABrB) or 3 days for
Colletolrichum. Growth of hyphae was both intercellular and intraceilular for both b g i
(Fig. 12B). A rating of 5 was obtaiaed for al1 areas of the endosperm within 2 days p s t -
contact for Cylindrocarpon and within 3 days pst-contact for Colletohichum. In
gened, Cylindrocapon produced more hyphae than Colletotrichum in al1 areas and at al1
days pst-contact (Fig 12C; Table 4).
HM* W W MEA PDA
Medium
HM HM* WM Mû+ MEA PDA WA
Meûium
Figure 1 1 . Average fungal colony diameters (mm) afier 2 weeks for A) Cylindmrpon and B) Colletotrichum on various media, with standard error bars. Media followed by an + indicates that plant growth regulators were added. Colony diameters with the same letter were not significantly different by the Student-Newman-Keuls test at P = 0.05 level of signifieance.
Figure 12. A) intact endosperm parenchyma cells in a control sarnple of dwarf mistletoe seed. B) Endosperm cells infected by Cylindmrpon at 7 days post-contact showing ce11 wall degradation, and both intercellular and intracellular colonization. x 200. C) Endosperm tissue colonized by Cylindrocarpo at 7 days post-contact. x 200.
The radicle of the seed was colonized l a s rapidly than the endosperm, and hyphae
were not observeû until day 3 for both fungi. Cell wall degradation was not evident until
7 days for Cylindmrpon or 14 days for Colletotrichum (Fig. 13A&B). Aggregates of
hyphae on the surface of the epiderrnis that penetrated into the ce11 layers below were
observed (Fig. 13C; Fig. 14C&D), which caused the epidennis to appear concave under
mechanical pressure (Fig. 14D). These fungal cushions were produced by both fungi, but
were only found in the rniddle or tip areas of the radicle. An acervulus was observed on
the epidennal surface of a radicle colonized by Colletotrichum (Fig. 13D). The tip was
heavily colonized by Cylindrocupon (Fig. 14A&B), with the maximum number of
hyphae in 1 mm2 being 2857. With Colletonichum, the top (area 1) never became
colonized, while the middle and tip areas attained ratings of 2 and 1, at 14 days post-
contact.
Growth of callus in the presence of Cylindrocarpon (Fig. 15) and Colletotrichum
(Fig. 16) was significantly reduced (P<O.OS) compared to uninoculated controls afier a
one-way ANOVA was done on callus diameter increase. Light rnicroscopic observations
revealed that Cylindroccirpon was more aggressive in colonization and cell walls were
degraded 0.5 days pst-contact (Fig. 17A&B). There was no cushion formation observed,
but both intercellular and intracellular hyphal growth was present. Maximum
colonization (ratin@) occurred at 1 day pst-contact for Cylindrocarpon and 3 days
post-contact for Colletottr'chum (Table 5). Celis infected by Cylindrocapn were
disorganizeâ., appeared plasmolysed, the cytoplasm was grainy, and nuclei were not
visible (Fig. 1 7C&D).
Table 4. Colonizatiïm of genninated seeds of dwarf mistletoe challenged with Cylindroca>pon and Colletotrichum as expressed by number of hyphe in 1 mm2 of tissue.
Days pst-contact Pathogen Location Areaa 1 2 3
Cylindrocarpon Endosperm 1 0 46 1 70
4 76 20 250 Radicle I O O 1
2 O O 49 3 O 2857
Coiïetotrichum Endospem 1 O O 117 2 O 1 185 3 O 1 202 4 1 1 1 45
Radicle 1 O O O 2 O O 1 3 O O 5
' Refer to Fig. 10
Figure 13. Dwarf mistletoe geminated seeds infected by Colletotricl~um. A) Intact cells of the control seed. Phase contrast, x 25. B) Cell wall degradation in infected seed at 14 days post-contact. Phase contrast, x 25. C) Colonized radicle tip at 3 days post-contact. x 100. D) An acervulus that developed on an infected radicle at 7 days post-contact. x 200.
Figure 14. Dwarf mistletoe radicles infected by Cylindrocarpoii. A) Colonized radicle tip at 3 days post-contact. x 40. B) Radicle tip shown in Fig. 5A at a higher magnification. x 100. C) Colonization of radicle at 3 days post-contact. x 100. D) Cushioning on radicle surface at 3 days post-contact. Note the concave nature of the epidermis. x 200.
O 2 4 6 8 10 12 14 16
Incubation time (days)
Figure 15. Growth of callus in the absence of (control, represented by open circles) and presence of (dual culture, represented by closed circles) of Cylindrocarpon.
incubation time (deys)
Figure 16. Growth of cailus in the absence of (control, represented by open circles) and presence of (dual culture, represented by closed circles) of Colletonichum.
Figure 17. Callus that has been colonized by Cylindrocarpon. A) Intact callus cells of the control sample. Phase contrast. x 200. B) Cell wall degradation in colonized callus at 3 days post-contact. Phase contrast, x 200. C) Intact callus cells of the control sample. x 200. D) Colonized callus cells at 0.5 days post-contact. Ce11 designated by anow is plasmolysed and the fwgus has penetrated intracellularly. x 200.
Table 5. Colonization of callus of dwarfmistletoe chdlenged with Cylindrocarpon and Colletotrichum as expressed b y ratings (0-5) and number of hyphae in 1 mm2 of tissue (in parentheses).
~ a ~ s post-contact . -
Pathogen Area 0.5 1 2 3 7 Cylindrocarpon 1 1(1) 5(95) 5(195) S(261) 5(390)
2 5(44) 5(130) 5(145) 5(270) S(121) Avg 3(23) 5(113) S(170) 5(265) 5(256)
Colletotrr'chum I O 3(18) 4(18) 5(160) 5(276) 2 O 3(2) 3(24) 5(264) 5(199)
Avg O 3(11) 3.5(21) 5(212) 5(23 8)
43. DISCUSSION
There are many advantages of using tissue culture to study host-pathogen
interactions. Host cens cm be challenged with the pathogen in the absence of
contaminating microorganisms or tissue injury, the environmental conditions can be
precisely controiled, inoculum levei can be regulated, preçursors and products cm be
added or removed easily (Ingram 1976) and cells that are undergohg infection with
pathogens can be closely observed (Ostry and Skilling 1992). In the case of western
hemlock dwarf mistletoe, a parasite of higher plants, tissue culture was the only approach
that could be used to study the infection process by two pathogenic funpi. Dual cultures
of calli with pathogens have been conducted with many host-pathogen systems, including
obligate parasites such as Plasmopara viticola (Motel 1948), Cronartium jùsifonne
(Jacobi 1982; Jacobi et al. 1982), and Cronartium ribicola (Harvey and Grashm 1969;
1970).
Prier to initiating dual cultures, it was necessary to determine the conditions
ind uencing fungal growth on agar media suitable for callus growth, such as concentration
of plant growth regulators, temperature, amount of inoculum and incubation the. Al1 of
these factors can influence fcungal growth on the callus tissue (Miller 1985). A successful
dual culture is one in which a balance is achieved i.e. the callus and fiingus should grow
without either of the two becoming dominant (Miller 1985). A moderate b g a l p w t h
rate is desirable to avoid rapid colonization. In this study, media that mpported moderate
fiuigal growth were selected and they differed with the two fun@ used. Resulîs indicated
that both fiingi were able to grow on media used for the tissue culture of dwarfmistletoe
and that plant growth regulators had no effect.
In this shidy, Cylindrocarpon was more aggxessive at colonization than
Colletohichum. Samples were colonized more quickly, there were more hyphae
produced, and degradation of ce11 walls occurred more quickly. in nature,
Cylindrocarpon is a canker b g u s that attacks swellings produced by dwarfmistletoe,
with subsequent attack of the endophytic system (Hawksworth and Wiens 1996). In this
experirnent, the use of callus may be appropriate since it provides a mode1 of dividing
cells similar to the tree cambium (Hendry et al. 1993). ColZetohichum is the most lethal
pathogen of dwarf mistletoe shoots and bexries (Hawksworth and Wiens 1996), and
inoculation experiments have show that it is also able to invade the endophytic system
(Parmeter et al. 1959). In order for Colletotrichum to infect a potential host, it uses a
strategy tenned intracellular hemibiotrophy to colonize host tissue. This strategy consists
of 3 phases: biotrophy, benign necrotrophy, and destructive necrotrophy (Skipp et al.
1995). Biotmphy occurs as the infection hypha entea the lumen of the host epidermal
ce11 and swells to form an infection vesicle and primary hypha. The host plasma
membrane becomes invaginated, but the host cytoplasm continues to exhibit streaming.
Benign necrotrophy is associated with a number of ultrastruchua1 and physiological
changes in the host ce11 inciuding an increase in cytoplasm mass, decrease in cytoplasm
density, dilation of endoplasmic reticulum and chloroplast lamellae, inaease in plasma
membrane penneability, cessation of cytoplasmic streaming, rupture of tonoplast and
plasma membrane, and cytoplasmic disorganization. Destructive necrotrophy is
associated with the production of ceIl wall degding enzymes, and the appearance,
expansion, and darkening of lesions as secondary hyphae develop as branches fiom the
primary myceliurn in dead cells. Ou results corroborate uiis pathogenesis process, which
may explain why ColIetonichum was slower in its ability to colonize seeds and callus
compared to Cylindmcarpon. Benign necrotrophy was seen with the aid of the light
microscope, witnessing a decrease in cytoplasm density , rupture of tonoplast and plasma
membrane, and cytoplasmic disorganization. The evidence of destructive necrotrophy
was seen, as ce11 walls were degraded by both fun@ used in this study.
Callus diameter was significantly reduced by inoculation with the two pathogens.
Similar results were found by Sieber et al (1990) when the endophytic pathogen
C-prodiaporthe hystrix inhibited Acer macrophyllum callus in dual culture. Callus has
been used successfully for in vitro screening of resistance to wood-destroying fungi (Hrib
and Rypacek 1983) and to coffee berry disease caused by Colletohichum kahawae
(Nyange et al. 1995). However, since callus lacks a cuticle and epidermis, which may be
necessary for fungal recognition and signaling, callus may not be the optimal explant in
host-pathogen studies . In tobacco infected with Phytophthora paraîirica var nicotianae,
shoots and compact tissues showed resistance whereas loose fiiable callus was
susceptible regardless of tissue genotype or fungal race (Helgeson 1983). Excess
nutrients on the soft, wet tissue provided a desirable foodbase through which resistance
was readily overcome (Helgeson 1983). Gemiinated mistletoe seeds are therefore the
preferred explant when developing in vitro screenhg methods since they more closely
resemble the intact plant, with a cuticle and e p i d d s . Howeva, the fact that
colonization of d u s did occur suggests that both gemiinated seeds and callus can be
used in an in vitro screening systern for potential biocontrol fun@ of dwarfmistletoe.
The method developed in this study was usehl to elucidate the host-pathogen
interactions of Cylindrocarpon cylindroides and Colletolrichum gloeosporioides and was
sensitive enough to show that there was a difference in aggressiveness between the two
fungi. Thus, this technique can be used for rapid differentiation of other potential
biocontrol fùngi. It is not known whether the h g i used in this study produced any
secondary metabolites that may be toxic to dwarfmistletoe. Future research could
involve plating dwarf mistletoe callus on media amended with culture filtrate, incorporate
bioassays to detect phytotoxic activity in the culture filtrate, and characterize the
phytotoxic component(s) of the culture filtrate.
5.0. CONCLUSIONS AND FUTURE RESEARCH
A comprehensive review of tissue culture of parasitic flowering plants was
conducted to provide cunent knowledge on the biology, distribution, host range, and
tissue culture procedures. Dwarfmistletoes (Arceuthobium spp.) are the most
evolutionarily specialized genera of Viscaceae and are destructive parasites of
commercially important forest trees. Dwarfrnistletoes weaken trees by slowly removing
water, minerais, and photosynthates, causing reductions in vigour, growth, seed
production, wood quality, and even host mortality. Control methods for dwarf mistletoe
include silvicultural, chernical, genetic, and biological control with fun@ and insects. A
new management tool is necessary as the change in forestry practice has lead to a
reduction in cut block size and a move to partial cutting operations. These operations
increase the ratio of edge trees to seedlings, thus enhancing mistletoe spread and damage.
In vitro cul^ of dwarf mistletoe is usehl to study anatomy and development,
dong with the host-parasite relationship. Physiological studies leading to the control of
this parasite can be also be investigated. The first report of in vitro culture of western
hemlock dwarf mistletoe (A. tsugense subsp. mgense) is describeci. A factorial
experiment evaluated the effects of media, tempera-, presence or absence of light, and
plant growth regulators on the production of radicles, holdfasts, and callus. Mistletoe
seeds gemiinated and holdfasts were fomed in culture, indicating that these stages were
not dependent upon contact with a living host. Optimal conditions for radicle elongation
were White's medium at 200C in the presence of light and without plant growth
regulators. Holdfasts developed fiom tips of radicles or tkom swollen radicles, and wae
maximally produced with Harvey's medium, light, 2,4-dichJorophenoxyacetic acid (2,4
D) at 1 mg 1-', and 6-benzylaminopurine (BAP) at 0.1 mg 1". Callus arose fiom split
radicles and holdfasts and optimal development was on White's medium with 0.5 mg 1-'
2,4D and 1 mg ï' BAP at 20°C in the dark. Split radicles with cailus seem to be unique
to western hemlock dwarf mistletoe dong with the swelling of radicles to form spherical
holdfasts.
The successfùl in vitro culture of western hemlock dwarf mistletoe provided a
means to screen two naturally occurring fun@, Nectria neomacrospora (anamorph
Cy lindrocarpon cyhdroides) and Colletotn*chum gloeosporioides, as biocontrol agents
against dwarf mistletoe. A suitable medium for dual challenge studies was found, and
germinated seeds and calli were challenged with these fun@ in vitro. The colonized
explants were examined histologicaily to determine the process of pathogenesis. Cushion
development, ce11 wall degradation, and intercellular and intracellular colonization were
evident for germinated seeds challenged with both fungi. Growth of dwarf mistletoe
callus was reduced by both fungi, and ce11 wall degradation dong with intercellular and
intracellular colonization was evident. Cells infected with Cylindrocarpon cylindroides
were disorganized and appeared plpsmolysed. The in viîro screening method was useful
to elucidate the host-pathogen interactions, and was sensitive enough to d e t d e that
Cylindrocarpon cylindroides was more aggressive at colonization than Colletotrichurn
gloeosporioides.
Rogress in the area of tissue culture of dwarf mistletoe provides additional
res~arch opportunities. In vitro culture can be used as tool to snidy genetic resistance,
and molecular techniques such as foreign gene insertion or effects of gene dimuption
through antisense technology can be used to study parasite behavior. Cultures of dwarf
mistletoe may also be used in studying the physiological and biochemical mechanisms of
the host-parasite interaction. As in vitro culture was useci effectively in a screening
system, other potential biocontrol fun@ may be screened in the same manner. It is not
known whether the fungi used in this study produce any secondary metabolites that may
be toxic to dwarfmistletoe, and fbtwe research could involve testing culture filtrates for
phytotoxicity.
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