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. 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