In vitro Propagation of Avaucavia cunninghamii and Other
Species of the Araucariaceae via Axillary Meristems
G. E. ~ u r r ws
o AB, D. D.
ole^*, R. J. ~aines' and D.
G. ~ i k l e s
A ~ ~ t aDepartment,
ny
University of Queensland, St Lucia, Qld 4067.
B ~ r e s e naddress:
t
School of Agriculture, Riverina Murray Institute
of Higher Education, P.O. Box 588, Wagga Wagga, N.S.W. 2650.
' ~ u e e n s l a n d Department of Forestry, Forestry Research Centre, M S . 483, Gympie, Qld 4570.
D ~ u e e n s l a n Department
d
of Forestry, Division of Technical Services,
P.O. Box 631, Indooroopilly, Qld 4068.
Abstract
Stem segments with 3-5 leaf axils, excised from the upper portion of the mainstem of 2-year-old hoop
pine (Araucaria cunninghamii Aiton ex D. Don) seedlings, produced orthotropic buds from the concealed axillary meristems when cultured on a basal medium (BM) of half-strength Murashige and Skoog
(MS) inorganic salts, the medium level of growth factors and amino acids of deFossard, 20 g L sucrose
and 6.5 g / L agar. This procedure was also successful with A . balansae, A. bidwillii, A. colurnnar~s,
A. hunsteinri, A. luxurians, A. montana, A. rule;, A. scopulorum and Agathis robusta and with stem
segments from orthotropic coppice shoots of juvenile morphology collected from the stumps of 20-yearold hoop pines felled near ground level. The hoop pine explants were highly sensitive to cytokinin; 1 PM
and 1 0 6-benzylaminopurine
~ ~
caused the formation of distorted buds and total inhibition of bud
development respectively. Lofier concentrations (0.001-0.1 + I ) did not noticeably influence bud formation or development. A low rate of multiplication was induced by reculturing the stem segments after
the excision of the initial shoots. New buds developed in the leaf axils of that part of the initial shoot
which remained attached to the primary stem explant.
Shoots derived from seedling and coppice cultures of hoop pine and seedling cultures of Agathis
robusta rooted in vitro on BM + 0.1-10.0 PM indole-3-butyric acid (IBA), but with only 5-20% success.
Up to 80% rooting was obtained if both hoop pine shoot types (i. e. from seedling and coppice cultures)
were cultured on modified BM (quarter strength MS salts, 10 PM IBA plus no agar) for 2 weeks, before
being transferred to a mixture of non-sterile peat and perlite or vermiculite and perlite, maintained under
a high humidity (90-95%). Plantlets were subsequently transferred to normal glasshouse conditions and
then to the field with less than 5% mortality. Thus hoop pine can be added to the relatively small
number of conifers for which the capacity to micropropagate juvenile and mature plants and successfully
establish their clones in the field has been demonstrated.
Introduction
The advantages of propagating trees in vifro have been well documented, as have the
benefits of propagating mature trees of proven performance, rather than embryonic or
seedling material (Bornman 1983; Selby and Harvey 1985). To date the use of embryonic explants has been common in gymnosperm micropropagation and, where material
from mature trees has been cultured, the initiation of adventitious buds (Jansson and
Bornman 1983; Bonga 1984; Chretien and Vieth 1985; von Arnold and Tillberg 1987)
rather than multiple axillary bud formation (Gupta and Durzan 1985, 1987; Abdullah
et al. 1986) has been the favoured pathway of organogenesis.
Araucaria cunninghamii Aiton ex D. Don (hoop pine) is a dominant component of
certain rainforests in eastern Australia and Papua New Guinea. It has been extensively
plan'ted on former rainforest sites in south-eastern Queensland and has potential for
G. E. Burrows et al.
tropical plantation forestry elsewhere; A. hunsteinii and A . angustifolia are also
important in plantation forestry, while A. araucana and A . heterophylla are valued in
ornamental horticulture (Nikles 1980).
The traditional methods of vegetatively propagating these species are particularly
slow as many, if not all, species of the Araucariaceae possess a strongly determined
orthotropic and plagiotropic bud system which restricts the taking of cuttings or grafting
material to the mainstem, if upright plants are required (Haines and deFossard 1977;
Haines and Nikles 1987b). However, this restriction on the source and amount of
material available for propagation is partially offset as the Araucariaceae possess axillary
meristems in their numerous apparently blank leaf axils (Burrows 1986, 1987). In other
conifers leaf axils of similar external appearance are usually devoid of specialised budforming tissues. The Araucariaceae are also unusual amongst the conifers in that they
retain the ability to form coppice or epicormic shoots of juvenile morphology when
mature trees are felled or girdled near ground level (Veillon 1980; Haines and Nikles
1987b). This characteristic offers the possibility to mass propagate superior mature trees
and thus capture additional genetic gains (Haines and Nikles 1987~).Micropropagation
techniques have the potential to make efficient use of the limited orthotropic bud
reserve.
Previous studies of the in vitro culture of the Araucariaceae have been reviewed by
Handro (1986), Maene and Debergh (1986) and Plant (1986). Initial investigations had
shown that adventitious buds could be initiated on hoop pine embryonic and newly
germinated seedling materials (Burrows 1983), as described for other gymnosperms. This
paper reports further developments, namely the in vitro propagation of hoop pine by
axillary bud formation on segments from the leaders of 2-year-old plants and from
orthotropic coppice shoots on the stumps of felled 20-year-old trees. Mainstem segments from seedlings of a further eight species of Araucaria and from Agathis robusta
were also cultured in vitro.
Materials and Methods
Pieces of mainstem, free of branches and about 9 cm in length, were cut from the upper half of
2-year-old Araucaria cunninghamii seedlings (400-500 mm height, 7-9 mm base diameter, Fig. 1; see
also fig.33, Burrows [1986]). Similar material was collected from A. balansae, A . bidwillii,
A. columnaris, A . hunsteinii, A . luxurians, A . montana, A. rulei, A. scopulorum and Agathis robusta.
In addition to this seedling material, orthotropic coppice shoots of juvenile morphology, 10-30 cm long,
were collected from the stumps of recently felled 20-year-old hoop pines. Material was judged to be
juvenile if it had linear, reflexed leaves, as opposed to broad up-curved leaves. (See Burrows 1986, figs
33 and 34 for further details regarding shoot maturity.) Shoots were collected five times in 12 months
from 58 stumps at the Queensland Department of Forestry's Imbil seed orchard and twice in 8 months
from 20 stumps which remained after routine thinning at Brooyar State Forest, south-east Queensland.
This material was subjected to the following disinfestation regime. The cut ends of the stem pieces
were sealed with molten paraffin wax, then placed for 15 min in running water, 5 min in 70% alcohol,
15 min in half strength commercial bleach (2.5% available chlorine) plus a droplet of Tween 20 and
finally given 3 washes in sterile, distilled water. The stem pieces were then cut into 6-7 mm long explants,
each with 3-5 leaves, and placed with their proximal ends embedded in the medium. The disinfestation
regime resulted in visible contamination rates of 20-35% and 0-5% with field- and glasshouse-grown
material respectively. Most contamination was from saprophytic fungi, especially Pestalotiopsis spp.
The rigorous disinfestation regime did not harm the explants as the stem surface is covered by a thick
cuticle and the axillary meristems are buried beneath the stem surface and are protected by localised
periderms (Burrows 1986, 1987).
In a second explant type, the phellem layers were peeled from the base of 2-year-old (Fig. 2) and
6-year-old hoop pines, then longitudinal slices of cortical tissue (2-5 mm in depth) which contained
buried axillary meristems (Burrows 1986) were excised and placed into culture. Beyond wiping the
surface of the stem with 70% alcohol, no disinfestation procedures were used as the removal of the
phellem exposed a sterile surface.
In vitro Propagation of Hoop Pine
The basal medium (BM), based on that of Haines and deFossard (1977), consisted of half strength
Murashige and Skoog (1962) (MS) mineral salts, the medium level of growth factors of deFossard (1981),
20 g ' L sucrose and 6.5 g 'L Difco Bacto agar. The medium was adjusted to pH 5.7, dispensed at 10 ml
per 76 mm x 25 mm polycarbonate tube, capped with polycarbonate screw-on lids and autoclaved at
121°C for 12 min at 103.4 kPa. Various organic substances [activated charcoal (AC), adenine sulfate]
and plant growth regulators [6-benzylaminopurine (BAP), 6-(y,y-dimethylallylamino)-purine (2iP),
gibberellic acid (GAJ), indole-3-butyric acid (IBA), kinetin (K), a-naphthaleneacetic acid (NAA)] were
added individually or in various combinations, as indicated.
Unless otherwise stated, cultures for both bud and root production were incubated at 25 f 2"C, under
a 16 h photoperiod, with a photon irradiance of 80-100 pmol m - 2 s - from cool white fluorescent tubes.
'
Results and Discussion
Bud Development: Seedling Material
Stem segments from 2-year-old hoop pines produced axillary buds when cultured on
BM (Fig. 3). These buds, which are derived from little-differentiated axillary meristems
(Burrows 1986), took 17-21 days to emerge in the leaf axils, but only a further 3 weeks
to reach 6-8 mm in length. The time taken for bud emergence is similar to that for
adventitious bud formation in some species (Horgan and Aitken 1981; von Arnold
1982) and illustrates that the little-differentiated axillary meristems require time to
undergo an ordered development. This procedure was successful with over 80% of the
several hundred seedlings cultured, although within each plant a morphogenetic gradient
existed so that explants excised in increasing proximity to the shoot apex displayed a
decreasing capacity for bud development. This may be related to a developmental stage
of the axillary meristems or correlative inhibition by the apical bud. Similar results have
been reported for A , heterophylla (Maene and Debergh 1986) and in other species, for
both axillary and adventitious budding (Boulay 1979a; Bressan et ai. 1982; Yu and
Meredith 1986). Consequently, explants were routinely excised from the most proximal
parts of the stem which did not have excessive secondary xylem development. Within
responsive explants all axillary meristems commenced development into buds, as indicated by the presence of swellings in the axils, but the lower ones were quickly inhibited
(Fig. 3). The uppermost buds formed a short mainstem followed by the development
of two or three vigorous branches (Fig. 7). Thus few new orthotropic axillary meristems
are formed relative to plagiotropic ones, which restricts the supply of suitable material
for the subsequent multiplication steps.
Axillary bud formation has also been reported for mainstem segments from
18-month-old A. cunninghamii (Haines and deFossard 1977), etiolated 30-day-old
A. angustifolia (Handro and Ferreira 1980), 70-year-old A. heterophylla (Maene and
Debergh 1986), 15-month-old A , heterophylla (Plant 1986) and branch segments from
20-year-old A. angustifolia (Handro 1986). In contrast to hoop pine the stem segments
of A. angustifolia appear to produce little branch material (Handro 1986, fig. 1C).
The addition of 0-001, 0.01 or 0 . 1 p~ BAP to BM had no significant effect on bud
development, while at 1 .O ,UM it caused the formation of distorted axillary buds of
limited growth and at 10 p~ it was totally inhibitory to bud development. BAP, at the
concentrations tested, did not induce the formation of adventitious buds, nor did it
promote the meristems in the lower axils to develop into vigorous buds. 2iP (0.01, 0.1,
1, 10 p ~ and
) adenine sulfate (50, 100, 200,400 p ~ also
) produced bud distortion at high
concentrations, while K (0.01, 0.1, 1 . 0 , 10 p ~ and
) NAA (0.01, 0 . 1 and 1 . 0 PM) had
no effect on bud initiation and growth. All GA3 concentrations tested (0.1, 1.0, 10,
100 ,UM)were inhibitory to bud development. In a similar manner, stem segments from
juvenile plants of A. heterophylla produced optimal bud development on hormone free
media (Plant 1986), while stem explants from mature plants of the same species were
not significantly influenced by K , BAP or 2iP (0.1-5-Omg/L) (L. Maene and
G. E. Burrows et al.
Fig. 1. Upper portion of the mainstem of an Araucaria cunninghamii seedling 2 years of age,
from which explants having 3-5 leaf axils were subsequently excised. Note that the leaf axils
are apparently devoid of any buds or bud-forming tissues. Note also that the two proximal
branches are separated by several leaves, while the three distal branches are the first to be
arranged in a pseudowhorl. These uppermost branches have almost overgrown the terminal
orthotropic shoot apex. Scale: 1 cm.
Fig. 2. Surface view of the base of the mainstem of an Araucaria cunninghamii seedling 2
years of age from which the phellem layers had been peeled away, exposing the phellogen.
The small, dark areas (arrowed) are the axillary meristems, while the white ovals indicate the
position of the former leaf bases. The distal end is to the top. Scale: 4 mm.
Fig. 3. Mainstem segment from a hoop pine 2 years of age after 5 weeks of in vitro culture
on BM, showing development of the axillary meristems into buds. Note the inhibition of the
lowermost bud. Scale: 4 mm.
Fig. 4. Mainstem segment from an Araucaria hunsteinii seedling 2 years of age after 5 weeks
o n BM. Note that some swelling has occurred in the lower two axils but buds have not
emerged. Scale: 4 mm.
In vitro Propagation of Hoop Pine
P . Debergh, personal communication). A relatively high concentration of K (2 mg / L)
was required for bud formation in stem segments of A . angustifolia (Handro and
Ferreira 1980).
1 or 10 y~ BAP formed obvious swellings in their axils 7
Stem explants on BM
to 10 days before those on BM. Pulsing experiments were undertaken to determine if
the early stimulatory effect of BAP could be used to increase bud growth or induce
multiple bud formation. Culturing explants on BM + 1 or 10 ,UM BAP for 1,2,3 or 4
weeks, before transferring them to BM, did not increase bud growth or the number of
vigorous buds. If explants were cultured on BM for 3 weeks, which allowed the axillary
meristems to develop into small buds, before transferring them to BM + 1.0 PM BAP,
bud elongation slowed and new buds developed in the axils of the recently initiated
leaves, thus inducing a type of multiple budding.
Investigation into the composition of BM, with respect to successful bud development, showed: (1) the various growth factors could be omitted, (2) half and threequarter strength MS mineral salts were not noticeably different from the medium level
of mineral salts of deFossard (1981) and these were better than quarter or full strength
MS mineral salts, (3) at least 15 g L - of sucrose was required, which was not noticeably
different to 20, 25 and 30 g L - I , and (4) culturing the explants on filter paper bridges
dipped in liquid BM did not support bud growth.
The addition of AC (0.1 and 1.0%) to BM promoted a slight increase in shoot
growth over 35 days, but after 4-5 months on unrenewed medium those explants on
BM + AC had 4-5 times the shoot growth of those on BM. These findings are consistent with numerous other studies where it was assumed that the AC had absorbed and
inactivated various inhibitors which had been released into the medium (Mehra-Paltra
et al. 1978; Boulay 1979b; von Arnold and Eriksson 1981; Pate1 and Thorpe 1984;
Rumary and Thorpe 1984), although the formation of abnormal shoots on media containing AC has been noted (von Arnold 1982).
Little research into optimal incubation temperatures for conifer tissue culture has
been performed and in the absence of such experimentation 22-25°C has been routinely
used. Increased incubation temperature for hoop pine stem segments produced an
almost linear increase in shoot growth. At 22°C shoots averaged 2.8 mm in length after
35 days in vitro; at 2S°C, 4 . 5 mm; at 28"C, 5.5 mm; while at 30°C shoots averaged
6 . 5 mm and exhibited no signs of abnormality. Embryos of Picea abies died when
cultured above 30°C and adventitious bud initiation on embryos was best between 20
and 25°C (von Arnold and Eriksson 1978). Similarly, the highest percentage of buds
of Picea abies forming adventitious buds was at 20°C (von Arnold and Eriksson 1979a);
however, in Pinus contorta 27°C was found to be better than 20°C for adventitious bud
initiation, although 22°C was better than 27°C for shoot elongation (Pate1 and Thorpe
1984). The higher optimal incubation temperature of hoop pine is probably related to
its tropical and subtropical origins as opposed to the temperate origins of most other
investigated conifers, although differences between preventitious and adventitious bud
development do not allow for direct comparison.
Stem explants of Araucaria balansae, A . bidwillii, A . columnark, A . hunsteinii
(Fig. 4), A . luxurians, A . montana, A . rulei, A . scopulorum and Agathis robusta
(Fig. 5) produced axillary buds in a manner similar to that described above for
Araucaria cunninghamii, when cultured on BM. The buds of Agathis robusta took up
to 6 weeks to emerge in the leaf axils as the axillary meristems are buried deeper in the
cortex than in the araucarias (Burrows 1987).
Likewise, the slices of hoop pine cortical tissue produced buds from the buried axillary meristems when cultured on BM or BM + 1.0% AC (Fig. 6). At present it is
necessary to fell or girdle mature trees near ground level to obtain juvenile coppice or
epicormic shoots, which is undesirable when dealing with superior genotypes. With this
technique it should be possible to excise a single meristem from the base of a tree, thus
+
'
G . E. Burrows et al.
I n vitro Propagation of Hoop Pine
causing minimal damage. This meristem could then be the source of all subsequent bud
multiplication, either axillary or adventitious.
Bud Formation: Coppice Materials
Two types of coppice shoots were produced on the 20-year-old hoop pine stumps
examined; one had a diameter of 4-5 mm with a well developed xylem cylinder, while
the other had a diameter of 7-8 mm with a wide cortex and little xylem development.
Bud formation on explants from the former coppice type, which was morphologically
and anatomically very similar t o the 2-year-old seedling material, was good, as over 80%
of the 78 genotypes tested formed vigorous'buds in vitro. Shoots could be successfully
collected and cultured all year, in contrast to some northern hemisphere conifers for
which a strong seasonal influence on adventitious bud initiation has been recorded (von
Arnold and Eriksson 19790; Gupta and Durzan 1987). Explants from the latter coppice
type either failed to produce buds or the buds that developed were of poor form and
slow growing. The similar morphology and performance of the seedling material and
the comparatively narrow coppice type suggest that most information gained from the
former can be directly applied to the latter.
Most conifers do not form juvenile coppice or epicormic shoots when mature trees
are girdled or felled, although Sequoia sempervirens, Taxodium distichum and
Cunninghamia lanceolata are well known exceptions (Lust and Mohammady 1973;
Boulay 1979~). Consequently considerable effort has been expended on rejuvenating
mature tissues so they are more responsive in vitro. In contrast, the pronounced coppicing ability of the Araucariaceae (Veillon 1980; Burrows 1987) should ensure that the
micropropagation of mature trees is relatively straightforward. The 'cortical slice' technique developed with hoop pine is an extension of the use of coppice shoots in that the
coppice-forming meristems are utilised but with relatively little tree damage. Attempts
have been made to rejuvenate A. heterophylla, without the use of coppice or epicormic
shoots, by applying foliar sprays of BAP and 'cascade-grafting' of plagiotropic branch
tips onto seedling stocks but with little success (Maene and Debergh 1986).
Multiplication
After excision of the primary shoots, the hoop pine stem segments, both seedling and
coppice, were recultured on BM and new buds developed in the leaf axils of that part
of the primary shoot which remained attached to the initial stem explant (Fig. 7). T o
increase the multiplication rate, the mainstem of each excised shoot was cut into segments 3-4 mm long, which were then split longitudinally and cultured on BM; 70-80070
of these segments quickly formed small buds in their leaf axils, but the buds did not
elongate into shoots. In most species once buds are formed they will continue to
elongate on hormone free media, although some workers have noted problems in achieving elongation of conifer buds in vitro (Bonga 1984). A variety of modifications to BM,
including the use of quarter and full strength MS salts; the low and high levels of
deFossard (1981) growth factors; 10 and 40 g L - ' sucrose, filter paper bridges dipped
in liquid medium and the addition of NAA (0.01, 0.1 ,UM) and BAP (0.01, 0.1, 1.0,
10 PM), did not improve bud elongation. The above media modifications were used
singly, except for the NAA and BAP which were used in combination. As GA3
(deFossard et al. 1978; Wochok and Sluis 1980) and AC (Pate1 and Thorpe 1984;
Rumary and Thorpe 1984; Mudge 1986) have been found to promote shoot elongation
the secondary stem segments were cultured on BM for 3 weeks to allow bud formation
to occur and were then transferred to BM plus 0.1, 1* 0or 10 PM GA3 or BM plus 0.1
or 1.0% AC. No further bud growth occurred at any GA3 concentration, while 1%
AC produced a slight increase in bud elongation. These results are unlike those of
previous studies in respect of the positive effect A C can have on shoot elongation (von
G . E. Burrows et al.
Arnold and Eriksson 1981; Rumary and Thorpe 1984; Mudge 1986), but are similar
to other research in that GA3 did not promote elongation (Pate1 and Thorpe 1984;
Mudge 1986). Likewise, numerous media compositions were tested on A. heterophylla
to induce axillary bud elongation but with little success (Maene and Debergh 1986).
Adventitious buds produced on female strobili of Larix decidua also failed to elongate
in vitro (Bonga 1984).
The fact that the same leaf axils produce vigorous shoots when attached to the
original stem explant (Fig. 7) but not when cultured independently indicates that the
parent explant supplies some hormonal or nutritional substance or substances not supplied by the media tested to date. The small size of the explants may also interfere with
nutrient uptake systems that require particular tissues, or sink effects in removing toxic
substances. Unless this problem is overcome, clone sizes will be small and will be
produced relatively slowly, although this will be an improvement on current vegetative
propagation procedures which make relatively inefficient use of the limited number of
orthotropic buds and meristems.
Rooting and Planting Out
A low level of success (5-20%) was obtained when attempting to root shoots from
hoop pine seedling and coppice cultures and Agathis robusta cultures in vitro on BM.
Modifications to BM including reduction of MS salts to quarter strength, reduction of
sucrose to 1% or the addition of 0.5-10 PM IBA or NAA did not improve rooting
percentages. When shoots were supported on filter paper bridges with their bases
immersed in liquid BM + 1.0-10 p~ IBA no rooting occurred as a large friable callus
formed around the shoot base after 4-5 weeks. However, when shoots were cultured
on liquid BM modified to have quarter strength MS salts and 10 PM IBA for 2 weeks
before being transferred to either a vermiculite/perlite (50150) mix or a peat1perlite
(60/40) mix, maintained under a high relative humidity, rooting percentages improved.
In one experiment 40% (24 out of 60) of shoots from 2-year-old cultures rooted after
4 months, while in another 71% (30 out of 42) of shoots from hoop pine coppice
cultures rooted after 8 months (Fig. 8). The resulting plantlets had a well developed
cuticle and were transferred to normal greenhouse conditions and subsequently to the
field with less than 10% mortality. Similar reports of improved rooting under nonsterile conditions have been reported for many species, including several conifers
(Mehra-Paltra et al. 1978; Boulay 1979a; Horgan and Aitken 1981; Bornman 1983;
Mudge 1986).
Hoop pine seedlings initially produce short branches, which have only a single order
of branching, in a rather random arrangement (Fig. 1). It is only when the plants are
approximately 30 cm in height and 1-2 years of age that they begin to produce the
pseudowhorls of large branches that are characteristic of the genus (Figs 1 and 9).
Plantlets derived from the upper half of the mainstem of 2-year-old hoop pines produced long branches arranged in pseudowhorls when they commenced growth in vivo
(Fig. 9). These first-formed branches were relatively weak in relation to their size and
consequently had a drooping habit (Fig. 9). Thus hoop pine seedlings and plantlets
exhibit clear morphological differences based on their developmental and ontogenetical
origins. The long, trailing, ground-level branches could be a problem in a nursery
situation but the early development of large vigorous branches appears to be correlated
with good initial growth rates.
The use of axillary buds and low or no exogenous growth regulator levels should
ensure that the genetic stability of the regenerated plants is comparable to plants produced by traditional vegetative means. This method also has the advantage of having
been successful with over 80Vo of the genotypes cultured. In several investigations
dealing with adventitious bud formation in conifer tissues a pronounced variability in
the percentage of explants forming buds a n d / o r the numbers of buds formed on each
In vitro Propagation of Hoop Pine
responsive explant has been reported (von Arnold and Eriksson 1979b; Mott and
Amerson 1981; von Arnold 1982; Bornman 1983; Ellis and Bilderback 1984; von
Arnold and Tillberg 1987). This variability is usually attributed to genotypic differences,
although in some cases it may be due to the physiological condition of the source
material, the type of explant excised and the cultural conditions. Variability is relatively
unimportant when dealing with embryonic material but it has the disadvantage that the
probability of being able to propagate a single selected superior genotype will be relatively low.
Few conifers are propagated in vitro via axillary meristems or buds, while in the
angiosperms enhanced axillary branching is a common multiplication procedure. This
contrast is largely related to differences in leaf axil anatomy. Most angiosperms possess
in each leaf axil a well developed but inhibited bud which, when released from apical
dominance, can elongate to form new leaves and hence new axillary buds. In most
conifers few axillary buds form relative to the large number of leaves (Burrows 1986),
thus adventitious bud initiation has been identified as the most viable method of proliferation (Jansson and Bornman 1981). In Pinus most leaf axils possess some type of
Fig. 9. Micropropagated plant derived
from the upper mainstem of a 2-yearold hoop pine. Note that the lower
branches are considerably more
developed than those of an equivalent
position in a seedling. Note also that
these branches are of a drooping habit
and it is not until the next whorl that
the branches make the typical angle
with the stem. Scale: 12 cm.
bud, either fully formed or a residual fascicular meristem, hence several species have
been propagated via these structures (Gupta and Durzan 1985; Abdullah et al. 1986;
Mudge 1986). In these species and other conifers propagated via axillary buds the rate
of multiplication appears to be slower than that recorded for most angiosperms. The
leaf axil anatomy of the Araucariaceae has been indicated as intermediate between that
of the other conifers and the angiosperms (Burrows 1986, 1987). This study indicates
that their response in vifro, in terms of bud proliferation, could also be considered
intermediate.
Hoop pine axillary meristems exhibit a high sensitivity to cytokinin level: 1 . 0 ~ L M
BAP or 2iP consistently produced the formation of distorted buds, while 10 VM BAP
or 2iP was totally inhibitory to bud formation. Most conifer studies, especially those
dealing with Pinus species, have reported that 5-50 ~ L MBAP or K was required to induce
preformed buds to elongate (David 1982; Abdullah et al. 1986; Mudge 1986), although
no exogenous growth regulators were needed for bud development in Pseudotsuga
rnenziesii and Pinus larnbertiana (Boulay 1979b; Gupta and Durzan 1985). In their
quiescent state hoop pine axillary meristems are relatively undifferentiated, possessing
G. E. Burrows et al.
neither leaf primordia nor a definite apical meristem (Burrows 1986). When stem segments were placed on BAP-supplemented media it appeared that the accelerated cell
division induced by the hormone interfered with ordered cellular development. If the
axillary meristems were allowed to develop into small buds on hormone-free media (BM)
and were then transferred to BM + 1 a0 p M BAP primary bud elongation slowed and
the axillary buds grew out. Thus, if the axillary meristems were allowed to develop into
buds before BAP application, a response more typical of the angiosperms was obtained.
The initial high sensitivity to cytokinin may be related to their undifferentiated state
when quiescent, but it is reduced once they have developed into buds.
Conclusions
The methodology to micropropagate hoop pines 2 years of age via the pre-existing
axillary meristems has been established. Information gained with this material appears
to be directly applicable to juvenile orthotropic coppice or epicormic shoots from hoop
pines up to at least 20 years of age, an age at which superior individuals can be positively
identified. While explant establishment, initial bud production and plantlet formation
are routine, the main drawback of the method is the low multiplication rate, which
currently restricts its use to special purpose applications where relatively low numbers
of true-to-type plants are required. Unless the problem of bud elongation can be overcome some form of adventitious bud initiation may be required to increase bud production. In this case careful monitoring of resulting plant performance would be needed
because while hoop pine root shoots are adventitious in origin and have an upright stem,
their general form is regarded as poor.
Acknowledgments
We thank the Queensland Department of Forestry for supplying the plant material
used in this study and the Pathology branch of the Department for the determination
of fungal contaminants. One of us (G. E. B.) acknowledges the financial support of a
Rural Credits Development Fund Studentship.
References
Abdullah, A. A,, Grace, J . , and Yeoman, M. M. (1986). Rapid micropropagation of Calabrian pine
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Manuscript received 10 August 1988, accepted 31 October 1988