Aust. For. Res., 1982, 12, 121-127

Propagation of Jarrah (Eucalyptus marginata) by Organ and Tissue Culture

I. 1. Bennett and 1. A. McComb

Environmental and Life Sciences, Murdoch University, Murdoch, W.A. 6150.

Abstract Micropropagation methods are described for the production of clonal lines from Eucalyptus marginata (jarrah) seedlings. Nodal explants from mature trees can also yield shoot cultures, but a high frequency of contamination occurs among such explants. Uncontaminated callus cultures can be produced from mature trees by culturing stamen filaments and shoots can subsequently be regenerated from this callus. The rooting percentage of shoot cultures from either nodes or stamen callus of mature trees is low compared with that from seedling explants. Considerable variation was observed between trees in the ability of stamen callus to regenerate shoots and in the frequency of rooting.

[O.D.C. 232.328 : 178.83 Eucalyptus marginata]

Introduction Jarrah, Eucalyptus marginata Donn. ex Sm., is an important timber tree in Western Australia (Beggs 1971) and there are no reports of success in conventional vegetative propagation from mature trees. Micropropagation techniques could possibly be used for jarrah because several papers report success for other Eucalyptus species (Hartney 1980; Hartney and Barker 1980). Although the ultimate aim of our work on jarrah was to use explants from mature trees, we first used seedlings as these are easy to sterilize and readily provide information on suitable media and conditions. This experience was then applied to explants from maturt'l trees. de Fossard and co-workers have successfully employed this strategy (de Fossard et al. 1977; Barker et al. 1977). Sections of cotyledon petiole provided callus with which shoot induction media were developed. These media were subsequently applied to callus from stamens from mature trees. Shoot proliferation media were tested on shoots from seedling material, and the appropriate media were then used for shoots regenerated from stamen callus and those from nodal explants of mature trees. Finally, methods for rooting shoots from seedling sources were tested. They were then used for shoots derived from mature trees.

Materials and Methods

Plant Material Seeds were provided by the Forests Department of Western Australia. Flower buds and nodal sections were collected from trees on Murdoch campus and from the forest in the Darling Range . • 122 I. J. Bennett and J. A. McComb

Seed Sterilization and Germination Seeds were rinsed in 90% ethanol for 30 s followed by 2% sodium hypochlorite for 20 min, then three rinses in sterile water after which the seed coat was removed and the embryo placed on moist sterile filter paper in Petri dishes. These were sealed with Parafilm and kept in the dark at 25°C. Between 90-100% sterility was obtained and 50-60% of seeds germinated after 16 days, with 45% being of a suitable size for the cotyledons and their petioles to be cut into 5-mm sections for callus induction. When shoot axes were required, the cotyledons and the roots were excised.

Sterilization of Flower Buds Jarrah flowers mainly between September and February. Buds of almost full size and as free as possible from insect and fungal damage were selected. They were soaked in 95% ethanol for 5 min, flamed, then treated with 2% sodium hypochlorite for 20 min. They were then rinsed thrice, cut longitudinally and the stamens removed and spread on the surface of the medium. Frequently, 100% sterility was achieved.

Sterilization ofNodal Sections Nodal sections 1 em long were cut from young clean stems of mature trees and the leaf cut off leaving a petiole of 0.5 em or less. The most successful explants were those with a young developing axillary bud c. 3 mm long. Explants were washed in running water for c. 1 h, then dipped in 80% ethanol for 30 s before treatment with 2% sodium hypochlorite for 10 min. After 3 washes, the basal end was trimmed and the stem inserted into the medium. Even with material apparently free from insect or fungal damage, contamination was usually high, and most of the uncontaminated shoots turned brown and died. Variation in the timing of the sterilization and the strength of the sodium hypochlorite did not improve this situation.

Media All media with the hormones added were dispensed in 10-ml aliquots in 8 x 2.5-cm screw-cap polycarbonate tubes and autoclaved for 45 min at 109°C.

Medium for Callus Induction from Sections of Cotyledon Petiole The medium consisted of Murashige and Skoog (1962) minerals, B5 vitamins and inositol levels from Gamborg et al. (1968) with 90 mM sucrose, 10 pM kinetin, 10 11M napthalene acetic acid (NAA), 10 g dm-3 agar, pH 5.7. Cultures were kept dark at 25°C.

Media for Shoot Induction from Cotyledon Callus After 4-5 weeks on callus induction media, hard yellow calli of 5-10 mm diameter had developed. These were transferred to media with the same minerals, vitamins, and inositol as above, plus 60 mM sucrose, zeatin (2.5, 5.0, and 10 pM) and indole acetic acid (IAA) (0.5, 1.0, 2.0 pM), pH 6.0. A similar medium has been used successfully by Lakshmi Sita (1979) to induce regeneration from lemon-scented gum calli. Cultures were placed in constant light (600-1000 lx at plant level) at 25°C. Each hormone level had 10 replicates and the experiment was repeated 3 times. The number of shoots produced after 6 weeks was recorded. Propagation of J arrah by Organ and Tissue Culture 123

Media for Shoot Induction from Stamen Callus An initially grey and, eventually, yellow lumpy callus produced by the stamen filaments was transferred to the same range of media described for the callus from the cotyledon petioles. later experiments with callus from stamens used only media with 0.5 .uM IAA and 10 .uM zeatin. Calli were transferred to new media every 4 weeks for 3-4 months.

Shoot Multiplication Media Two basal media were tested, the first being the multiplication medium described by Hartney and Barker (1980) which was developed by de Fossard et al. (1977) for E. ficifolia and E. grandis. Using de Fossard's broad spectrum terminology (de Fossard et al. 1974a), the medium consisted of medium levels of minerals, 10 .uM indole butyric acid (IBA), medium levels of cytokinins (1.5 .uM benzyl amino purine (BAP), and 1.5 .uM ldnetin) and high levels of growth factors and amino acids. Barker et al. (1977) found that shoot growth was improved by reducing the concentration of IBA from 10 to 5,uM. Hartney and Barker (1980) used this medium for 10 species of eucalypt and obtained at least a three-fold increase in shoot numbers in 3 weeks. A second medium was tested, based on the results of l.akshmi Sita and Vaidyanathan (1979). This was Murashige and Skoog minerals, vitamins and inositol (no glycine or casein hydrolysate) (M & S), 60 mM sucrose, 10 g dm3 agar and a range of BAP (2.5, 5.0, 10.0 .uM) and NAA (2.5, 5.0, 10.0 .uM), pH 5.8. Shoots were excised from seedlings or from callus and were inserted into the media. Ten replicates were used per treatment and the number of new shoots were counted after 4 weeks.

Media for Shoot Induction and Multiplication from Nodal Pieces Sterilized nodal sections were placed directly on the shoot multiplication media developed from the above experiments; i.e. with hormone levels BAP 2.5 ,uM, NAA 2.5 ,uM.

Media for Root Induction Hartney and Barker (1980) developed a rooting medium successful for various species, which was based on the de Fossard broad spectrum medium. It was approximately half strength M & S minerals, 60 mM sucrose, 8 g dm3 agar, pH 5.5. This medium was tested with a range of IBA and NAA concentrations. Rooting was also examined in full strength M & S minerals with B5 vitamins and inositol, 60 mM sucrose, 8 g dm- 3 agar and a range of IBA concentrations. All rooting trials had 25 or more explants per treatment and were scored after 3 weeks. Cultures were initially placed in the dark for 7 days, then into a 16 h light/8 h dark regime (Franclet and Boulay 1982).

Transfer of Plantlets to Soil Plantlets were removed from tubes and as much agar as possible dislodged without damaging the roots. They were planted in seed trays containing a sterile 3:1 sand:peat mixture. The tray was placed under mist in a glasshouse. Humidity was gradually reduced over a period of 4 weeks by which time the plants had produced at least two new leaves. They were then potted individually and transferred to normal glasshouse conditions.

lllllllliii.__ 124 I. J. Bennett and J. A. McComb

Results

Callus Induction and Shoot Regeneration A fast-growing, hard, yellow callus was readily grown from the cotyledon and petiole sections. On transfer to the shoot induction medium and exposure to light, some areas turned green or red, and it was from these that shoots eventually arose. Media containing IAA 0.5 .uM and zeatin 10 .uM proved suitable for shoot induction (Table 1).

Table 1. Shoot production from jarrah cotyledon callus after 6 weeks culture Total number of shoots from 30 explants at each hormone level is shown; each explant is from a seedling from open pollinated seed. IAA (J.LM) 0.5 1.0 2.0

Zeatin 2.5 0 0 (J.LM) 5.0 10 2 3 10.0 22 6 5

Stamen callus placed on shoot induction media and exposed to light turned brown but also produced green patches. It was necessary to transfer the calli to fresh media several times before shoots appeared from the dark green areas. As with seedling callus, the best hormone levels were IAA 0.5 .uM, zeatin 5 to 10 .uM (Table 2).

Table 2. Number of jarrah stamen calli that produced green callus or shoots Ten explants at each hormone level. IAA (J.LM) 0.5 1.0 2.0

Zeatin 2.5 2* 1* (J.LM) 5.0 2* 1* 10.0 4 4 * Indicates those levels in which number of green calli is significantly different from that recorded on 0.5 J.LM IAA and 10.0 J.LM zeatin (P < 0.05) (chi square test). + Indicates those levels at which shoot primordia or shoots eventually developed.

Stamen callus from all trees tested produced green areas when treated as described above, but there was great variability between trees in ability to produce shoots. Three trees out of 30 tested gave results comparable to those reported in Table 2, the remainder did not produce shoots.

Shoot Production from Nodes ofMature Trees When sterile, living, nodal sections were obtained, a callus frequently grew from the region of the axillary bud and the cut ends of the petiole and stem. Some, however, produced vigorous shoots from the growth of the young axillary bud initially present. The percentage of explants (as counted before sterilization), that eventually gave success­ fu1 shoot cultures varied from 0-10% with different trees. However, the high level of initial contamination from most material and the callus production remains a problem.

Shoot Multiplication The medium developed by Lakshmi Sita and Vaidyanathan (1979), gave best growth Propagation of Jarrah by Organ and Tissue Culture 125

of shoots over the 4-week period of cultures. In the de Fossard medium used by Hartney and Barker (1980) the shoots deteriorated after 3 weeks. All results for shoot multi­ plication have been determined on M & S medium. The shoots from seedling axes and those regenerated from seedling callus showed similar mean multiplication rates of c. x10 on media with 2.5 pM BAP and 2.5 pM NAA (Table 3). Individual clones varied considerably giving rates between x3 and x20 during a 4-week period. Shoots from mature nodes and those regenerated from stamen callus generally showed a lower average multiplication rate (x3) and individual trees ranged from x2 to xS.

Table 3. Multiplication rates of jarrah shoots with a range of hormone levels after 4 weeks a. Shoots derived from seedling axes. Ten seedlings were tested. Nine shoots from each were produced and placed one on each medium. The mean number of new shoots is shown. NAA (JLM) 2.5 5.0 10.0 2.5 10.7 9.9 7.8* BAP 5.0 5.6* 7.7* 6.8* (JLM) 10.0 1.7* 3.5* 1.5*

b. Shoots derived from those produced on cotyledon callus. Shoots from 4 different calli were used. Ninety shoots of each were produced and 10 placed on each medium. The mean number of new shoots from all 4 lines is shown. NAA (JLM) 2.5 5.0 10.0 2.5 10.8 8.8 6.0* BAP 5.0 7.8 5.4* 7.2* (JLM) 10.0 2.2* 4.2* 2.8* * Indicates those rates significantly different from that rate recorded on media with 2.5 JLM BAP and 2.5 J.tM NAA (P < 0.05) (chi square test).

Root Production An initial experiment using Hartney and Barker's (1980) rooting medium showed that there was no difference between shoots originating from cultured seedling shoot axes and those regenerated from cotyledon callus - both showed 35% rooting at 5 pM IBA. Testing with a range of IBA and NAA concentrations (1.25-1 OpM) did not improve this percentage. However, a medium with full M & S minerals, and 60 pM sucrose with 10 pM or 15 pM IBA gave 4 7-52% rooting and the shoots remained green and healthy, though callus developed at the base. The rooting frequency of clonal lines from different seed­ lings was from 5% to 80%. Shoots derived from mature nodes and those from stamen callus were also rooted in M & S with 10 pM IBA. They showed a mean of 5% rooting with a range of 2-20% for clones from different trees. Several experiments unsuccessfully attempted to improve the rooting of shoots from mature explants by varying the hormones and their concentrations. Inserting the shoots up-side-down in the medium which promoted rooting in apple (Abbott and Wheatley 1976) was unsuccessful. Gupta et al. (1981) reported an improvement in the rooting of E citriodora after 3 subcultures of 60 days each. Some shoot cultures of jarrah originating 126 I. J. Bennett and J. A. McComb

from mature explants have now been in culture for a year with no marked improvement in rooting frequency.

Transfer to Soil The survival of different batches of plants varied, depending on whether they originated from seedling explants or mature trees, but even for the latter, survival was never less than 50%.

Discussion Micropropagation methods have been developed for jarrah using explants either from seedlings or from mature trees. Variability between mature trees makes it easier to apply micropropagation methods to some trees than others. This variability is also seen in seedling material but it is generally far easier to use seedling explants. The literature contains several reports of successful micropropagation from seedling material of other eucalypts (e.g. Hartney and Barker 1980) and a recent report of embryoids from seedling explants (Ouyang eta!. 1980, 1981). The cloning of eucalypts from seedlings is useful where valuable hybrids or mutants are produced, but the use of explants from mature trees has greater potential. Other eucalypts that have been cloned by organ culture from mature trees include E. grandis (Cresswell and Nitsch 1975), E. ficifolia (de Fossard 1978; Barker et al. 1977) and E. citriodora (Gupta et al. 1981 ). Reference is frequently made in the literature to the contamination problem experienced with mature trees. Use of flower buds allows abundant sterile material to be cultured, although there is the problem that buds are only seasonally available, and trees differ in their ability to regenerate shoots. However, previous. attempts to regenerate whole plants from eucalyptus callus from either seed­ lings or trees have largely been unsuccessful or resulted only in root formation (de Fossard 1974; Lee and de Fossard 1974; de Fossard et al. 1974b). Plantlet regeneration has been reported in only 3 instances: from E. alba hypocotyl (Kithara and Caldas 1975) and E. citriodora lignotubers and cotyledons (Aneja and Atal1969; lakshmi Sita 1979). The regeneration of shoots from jarrah anther callus reported here is further evidence that floral tissues may perform differently in culture or regenerate plants more readily than cultures from vegetative parts ofmature plants (Tran Thanh Van 1973; Vardi eta!. 1975; Radojevic 1979). Other workers have found difficulty in rooting shoots originating from nodal cultures of mature eucalypt trees, while cultures from seedlings of the same species root readily, and the difference may be attributable to the levels of a rooting inhibitor present in adult tissues (Paton et a!. 1970). It is of interest that the shoots regenerated from stamen callus showed the same low rooting frequency as shoots from nodes of these mature plants. In other species tissue cultured plants from mature explants may be physiologically different from seedlings or plants micropropagated from juvenile explants (Banks 1979).

Acknowledgments We gratefully acknowledge financial support from the Australian Reserve Bank and technical assistance by Ms Marie .Slade. Ian Bennett holds a Commonwealth Post­ graduate Award. 127 Propagation of J arrah by Organ and Tissue Culture

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