Planta (Berl.) 125, 87--90 (1975) 9 by Springer-Verlag 1975

Short Communication Organ Culture of Eucalyptus grandis L. R h o n d a Cresswell a n d Colette Nitsch Laboratoire de Physiologie Pluricellulaire, C.N.R.S., F-91190 Gif-sur-Yvette, :France Received 28 March, accepted 12 May 1975

Summary. Organ culture of nodes of Eucalyptus grandis permitted the rooting of nodes from much beyond No. 14, the highest node from which rooting has been described so far, namely up to node No. 80, the eotyledonary node being No. 1. Plantlets which were obtained from node 50 have been successfully transferred to the field. The classical methods of vegetative p r o p a g a t i o n are n o t successful when used on adult-phase trees of most species of Eucalyptus. B y the time t h a t these trees have reached the age a n d size a t which t h e y can be evaluated b y foresters a n d geneticists, t h e y have passed the stage a t which t h e y can be p r o p a g a t e d vegetatively. P a t o n et al. (1970) a n d Nicholls st al. (1970) have isolated from a d u l t tissue of E. grandis, three rooting inhibitors the c o n c e n t r a t i o n of which irmreases with the decreased rooting a b i l i t y of stem cuttings. These authors s t a t e 4 h a t in E. grandis there is a complete loss of rooting a b i l i t y when the base of the c u t t i n g is t a k e n above node No. 14 (The cotyledonary node being node No. 1). The techniques of organ culture in vitro were adopted i n a n a t t e m p t at obt a i n i n g rooting a n d t h e n plantlets which can be transferred to the field, from nodes above No. 14 i n E. grandis. Plant tissue for all experiments was taken from E. grandis trees grown from seed planted May 1972 and from plants regenerated from nodes from these trees. The plants were grown under various environmental conditions of the Phytotron at Gif sur Yvette. Branches (always above node 14) were excised from the tree and the cut ends sealed with molten paraffin wax. Surface sterilisation of whole branches consisted of a quick rinse in 70% ethanol containing severals drops of soap, followed by treatment in 7 % calcium-hypochlorite solution, 5 min for glasshouse grown specimens, 10 rain for field grown specimens, and by 3 rinses with sterile distilled water to remove all traces of the sterilising solution. The time of sterilisation must be reduced to 3 min with young tissue. Tissue thus sterilised was dissected between sterile white paper. It has been found that for the subsequent development of the axillary buds and initiation of roots, the most suitable nodal explant consisted of 1.0 cm of main stem of a diameter between 0.15 and 0.4 cm with at least half of the petiole included (Fig. 1). The dissected nodal explants are placed in a Petri dish with sterile distilled water for at least 2 h before planting them upright in the medium (Table 1). The cultures are placed in darkness at 28~ for 8 days and then in diffuse light (ca. 1500 lx). The d e v e l o p m e n t of a node in culture begins with abscision of the petiole before there is a n y obvious d e v e l o p m e n t of basal callus or axillary bud. After a basal callus has been formed there is either d e v e l o p m e n t of the axillary b u d or root i n i t i a t i o n from the callus. Roots begin to appear at 16 days a n d m a y c o n t i n u e to appear u p to 3 m o u t h s later. The roots i n i t i a t e d d u r i n g the first 6 weeks of culture

88

R. Cresswell and C. Nitsch

Fig. 1. Node at beginning of culture ( x 0) Fig. 2. Plantlet developed from node just before transfer to greenhouse, 12 weeks (x0) Fig. 3. Plantlets, age 6 months (reduced 10 times) Fig. 4. Plant developed from node 50 (parent plant 7 months old) which was successfully transferred to the field (13 months old) (reduced 20 times) arise within the basal callus ; after this, there is sporadic root development directly from the stem tissue, generally from the level of the node. Thus after 3 months of culture the nodes m a y have any one of the following aspects: basal callus only, axillary bud development but no root, roots from basal callus but no bud, or a small plantlet consisting of the developing axi]lary bud and a root formed at the base (Fig. 2). The percentage of plantlets developed from nodes above node 14 of the parent plant, varies but is generally about 30%. These plantlets can be successfully transferred to pots of vermiculite in the greenhouse if watered regularly with the nutrient solution of the Gif-sur-Yvette phy~otron (J.P. Nitsch, 1968) and protected from dehydratation for the first 15 days by a plastic bag. Fig. 3 shows 2 such plants developed from nodes after 3 months in the greenhouse while Fig. 4 shows a plant (13 months old) developed from node 50 after 7 months in the field at Gif-sur-Yvette.

Organ Culture of EucalyTtus grandis L.

89

Table 1. Constituents of medium (in molarity except where otherwise stated) On(NO3)2 KNOs KH2P04 MgSO~ FeSO~. 7 H~O Na2EDTA HsB0 8 MllS04"4 HsO ZnS04" 4 H20 CuS04" 5 HsO Na2MoO4. 2 H20

2.1 • 10-8 1.2 X 10-8 9.2 • 10.4 5.1 • 10-4 1.0 • 10.4 1.0 • 10.4 1.6 • 10.4 1.1 • 10.4 4.3 • 10-5 1.0 • 10-7 1.0 • 10 -5

Inositol Nicotinic Acid Pyridoxine. HC1 Thiamine.HC1 Glycine Biotin

5.6 • 4.1 • 2.4 • 1.5 • 2.7 • 2.0 •

10-4 10-5 10-8 10-8 10-5 10-~

Folic Acid

1.1 • 10 -e

Sucrose Indole-3-butyric acid Agar

6.0 • 10-2 1.0 • 10-5 8 gm/1

pH 5.8

The initial problem encountered with the E. grandis cultures was the presence of a brown exudate arising from the cut tissue; this exudate seemed to lead to the subsequent death of the organ or tissue culture. The amount of exudate increases with age and diameter of the stem, but it has been overcome to some extent in the organ cultures if the explant are firstly soaked in distilled water and secondly if the cultures are started in darkness for a period of at least 7 days. Soaking of the explants for at least 2 h in sterile distilled water prevents dessication of the tissue before planting, and more importantly, allows for the leaching of substances which are evidently inhibitory to the future development of the explant. Paton et al. (1970) have developed a bioassay for root inhibitory substances in E. grandis b y using water in which E. grandis stems were soaked. Nicholls et al. (1970) have shown that the three root inhibitors present in E. grandis decompose to inactive substances in water. Thus, although we have so far not made any studies of the water used to soak the tissue, we assume that the soaking resulted in elimination of the rooting inhibitors. The soaking also allows for the leaching of the phenolic substances which are a major problem with the culture of this species. After 2 h, water which contained 60 nodes was yellowish in colour and the pH had changed from 5.6 to 6.1. I t has also been observed that the nodal explant must contain at least half of the petiole but, contrary to the common practices for cuttings, preferably not with the leaf or part of the leaf attached. Presence of the leaf blade appears to inhibit root initiation and to increase the amount of exudate formed whereas complete elimination of the petiole prevents axillary bud development, perhaps as a result of damage to the bud meristem. However, although a part of the petiole must be present at the beginning of culture, root, bud or callus development is generally not observed until the petiole has started to abscise. If the nodal explant contains a small axillary bud at the time of planting, this develops at the same time as or immediately after the appearance of the root. Otherwise, an accessory bud can develop from within the node after about 20 days of culture. One of the problems encountered at the moment is that root initiation and development is not always accompanied by axillary bud development, and that sometimes an apparently healthy, developed axillary bud will die. This could 7

Planta (Berl.), Vol. 125

90

R. Cresswell and C. Nitsch

be the result of lack of or poor vascular connection between the root, which is generally embedded in the callus, and the axillary bud. Some of the factors found to influence root initiation in nodes of E. grandis are: season, age, and environmental conditions of the parent plant (the best conditions for the parent plant are 22 ~ and 12 h of high intensity light), position of the node on the parent plant (center part of the main stem; for lateral branches, nodes near the trunk are better than nodes further on the side branch), as well as conditions of culture such as light and growth substances in the medium. One of the problems now encountered is the distinct seasonal effect in form of a total inhibition of rooting in late winter and a spontaneous bud development in spring. Although there are still m a n y problems to be solved, this technique of organ culture by-passes the No. 14 node barrier of inhibition to rooting in E. grandis as observed by Paton and co-workers using cuttings. I t provides an effective means of clonal propagation of adult trees which is particularly important for E. grandis.

References Nicholls, W., Crow, W.D., Paton, D.M. : Chemistry and physiology of rooting inhibitors in adult tissue of Eucalyptus grandis. In: Plant growth substances 1970, p. Carr, D.J .ed. Berlin-Heidelberg-New York: Springer 1972 Nitsch, C., Nitsch, J.P. : The induction of flowering in vitro in stem segments of Plumbago indica L. I. The production of vegetative buds. Planta (Berl.) 72, 355-370 (1967) Nitsch, J.P.: Les Phytotrons et la bioelimatologie exp6rimentale. In: Encyclopedie Pl~iade, vol. Physiologie, p. 1602-1617. Paris: Gallimard 1968 Paton, D.M., Willing, R.R., Nicholls, W., Pryor, L.D.: Rooting of stem cuttings of Eucalyptus: a rooting inhibitor in adult tissue. Aust. J. Bot. 18, 175-183, 1970

Organ culture of Eucalyptus grandis L.

Organ culture of nodes of Eucalyptus grandis permitted the rooting of nodes from much beyond No. 14, the highest node from which rooting has been desc...
638KB Sizes 3 Downloads 0 Views