Plant Cell, Tissue and Organ Culture 15:233-244 (1988) Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands
Micropropagation of Alnus cordata (Loisel.) Loisel.
M. BARGHCHI Plant Physiology Division, Department of Scientific and Industrial Research ( DSIR), Private Bag, Palmerston North, New Zealand
Received 22 September 1987; accepted in revised form 27 July 1988
Key words: Alnus glutinosa, Italian alder, in vitro, micropropagation, sugars
Abstract. Procedures were developed for micropropagation of Alnus cordata through in vitro axillary shoot multiplication of axillary bud explants cultured in Murashige & Skoog (MS) medium. Establishment of cultures from plants grown in the field was very difficult due to bacterial contamination and phenolic oxidation in explants causing severe browning. Explants were first cultured on an MS medium containing 4.4#M 6-benzyladenine and 87.6mM sucrose (initiation medium) for 7 days and then transferred to an MS medium containing 1.1 ItM 6-benzyladenine and 333mM glucose (multiplication medium) for a further 20-25 days. It was necessary to transfer cultures from initiation medium to multiplication medium after 7 days to minimize excessive callus growth, abnormally thick and brittle leaves, inhibition of shoot elongation, and senescence. Shoot multiplication comparable to the above method was achieved by culture of axillary bud explants in MS medium supplemented with 1.1-4.4 #M 6-benzyladenine and 333 mM glucose 4--5 weeks after culture. Shoots rooted in MS medium (1/2 x macro-nutrients) supplemented with 1.24.9/tM indolebutyric acid. Also, 98% root- ing was achieved when cultures were treated with 625 mg 1 ~ indolebutyric acid for 24 h at the end of the shoot production stage and rooted in vivo as mini-cuttings. Plantlets established well in soil.
Alnus species are nitrogen-fixing actinorhizal plants that are particularly valuable in forestry and reforestation, land reclamation and conservation, revegetation of industrial mine spoils, landscaping, horticultural shelter belts and farm windbreaks, and biomass production. Clonal propagation of superior improved cultivars is essential to establish a uniform and high quality plantation. Clonal propagation of Alnus species from mature plants has been difficult, time consuming and of limited success. Procedures for micropropagation of some Alnus species have recently been reported [6, 11, 14, 15] as an alternative to conventional clonal propagation.
A. glutinosa (black alder) has performed especially well in horticultural shelter belts and farm windbreaks in New Zealand. A. cordata (Italian alder)
has proved to be marginally superior to A. glutinosa (the most widely used Alnus species) in horticultural shelter belts in New Zealand. A. cordata tolerates moderate summer drought in its natural habitat, is not subject to high infestation of aphids, and provides an extra fortnight of effective shelter at each end of the season .
A cordata is one of the more difficult species of Alnus to propagate conventionally. Micropropagation of A. cordata has not been reported before and this paper presents procedures for micropropagation from cut- ting-grown 3-year-old A. cordata originating from mature mother plants.
Materials and methods
Dormant axillary (5-10mm) bus and meristem tip (1-2mm) explants from 8-year-old mature trees of A. cordata (Accession Number 1736, supplied by Soil Conservation Centre, Aokautere, New Zealand) or axillary bud explants from rooted cuttings originating from the mature plants were used in this study. Hardwood cuttings were rooted in a cold frame in a rooting medium (1/2 pumice: 1/4 perlite: 1/4 peat) heated to 20 C. The base of the cuttings were dipped in a commercial rooting powder containing 8gkg -1 indolebutyric acid (IBA; mol. wt 203.2) (Seradix No 3, May & Baker) to promote rooting. Rooted stem cuttings at their third year of growth were used as experimental material. They were maintained in a glasshouse ventilated at 25 C and were pruned annually (3-4 times).
Axillary bud explants were surface-sterilized in 20% sodium hypochlorite (available chlorine is 5%) containing 0.1% wetting agent (Multi-Film X-77, Ivon Watkins-Dow Ltd) for 10min, followed by three rinses with sterile distilled water prior to culture in the media. Murashige & Skoog mineral salts and vitamins (MS)  containing 30 g1-1 sucrose (mol. wt -- 342.3), unless stated otherwise, was used as culture medium, which was gelled with 7 g l-1 agar (Davis Gelatine NZ Ltd).
The pH of the medium was adjusted to 5.6. Twenty ml of media was dispensed into 100-ml glass jars (covered with metal caps) and sterilized at 1.05kgcm -2 (121C) for 15min. Cultures were incubated at 26 _+ lC under 16h photoperiod at 20-25pEm-2s -~ produced from cool-white fluorescent lights.
Sterilized explants were incubated on MS media supplemented with the following plant growth regulators for 30 days: 6-benzyladenine (BA, at 1. l, 4.4, 17.6 or 35.2pM) or 6-furfurylaminopurine (kinetin, at 1.2, 4.6, 18.6 or 37.2pM) with all the possible combinations with naphthaleneacetic acid (NAA) at 0, 1.3 or 5.2 pM. Alternatively, cultures were initially incubated
in MS medium containing 4.4#M BA and 87.6mM sucrose (initiation medium) for 7 days and then transferred to shoot multiplication media. Shoot multiplication medium consisted of 87.6 mM sucrose and was sup- plemented with the following: BA (1.1 and 4.4#M), or 1.1#M BA plus either 0.3#M gibberellic acid GA3) or 3g1-1 activated charcoal (Darco Corp.). Cultures were incubated in 16 h photoperiod or in the dark. Shoot multiplication in MS medium containing 1.1/~M BA and 333 mM glucose, in 16 h photoperiod or in the dark, was studied too.
All shoots greater than 3 mm were counted and measured. The average shoot number and average shoot length per cultured explant were computed in analysing the results.
Shoots produced in MS medium containing 1.1 #M BA and 333mM glucose were cut as mini-cuttings (15-20mm) and were cultured in agar- solidified medium (1/2 macronutrients) supplemented with 1.2, 4.9 or 12.3 #M IBA to root. Rooting media contained 87.6 mM sucrose or 333 mM glucose. Some of the cultures in the medium containing 4.9 #M IBA and 333 mM glucose were either maintained in this medium throughout the rooting treatment or were transferred to a medium without IBA 10 days later; these cultures were either maintained in 16 h photoperiod or were kept in the dark for the initial 6 days of rooting treatment.
In order to reduce production cost, liquid MS medium containing IBA (0.025, 0.123, 0.615 or 3.076mM) was used to promote in vivo rooting of shoots. The medium was poured into the jars covering 10 mm of the base of the shoots at the end of the shoot multiplication stage under non-aseptic conditions. Shoots were kept in the liquid medium for 24 h and then cut into 15-20mm pieces and used as mini-cuttings to root in the potting mix. Rooting of mini-cuttings and establishment of in vitro produced plantlets were initially carried out on a propagation bench with high humidity (equip- ped with a fog unit) to prevent dehydration. After the pIants were establish- ed the humidity was reduced gradually over a period of 10-15 days.
A minimum of 15 replications per treatment was used throughout this study. Effect of treatments were analysed using analysis of variance and mean separations were calculated by Fisher's LSD (least significant dif- ference) test.
Results and discussion
Establishment of cultures from dormant axillary bud explants taken directly from trees in the field was very difficult due to contamination (mainly bacterial) and phenolic oxidation in explants causing severe browning.
Fig. 1. Inhibition of growth ofA. cordata cultures in culture initiation medium (MS + 4.4/~M BA + 87.6 mM sucrose) due to callus growth, and inhibition of shoot elongation (2 weeks after culture initiation).
Sterile cultures were established from 80% of the shoot tip explants dissec- ted from sterilized dormant axillary buds taken directly from trees in the field; however, their growth was not satisfactory as they had severe vitrifica- tion and died later. The dissection of meristem tip explants from the dor- 'mant axillary bud explants grown in the field revealed that the failure to establish sterile cultures was mainly due to the infestation of axillary buds with insects and insect larvae.
Of the hardwood stem cuttings taken into the glasshouse, 40% rooted. Explants from the actively growing shoots of these rooted cuttings seemed to be more suitable for culture establishment and were subsequently used throughout this study.
Culture establishment was initially satisfactory in media containing up to 17.6pM BA or 18.6/~M kinetin without any NAA. The cultured explants deteriorated within 2-3 weeks showing excessive callus growth, abnormally large and brittle leaves, inhibition of shoot multiplication and elongation, and senescence (Fig. 1). Subculture of explants to the same media did not improve the growth. Explants establish better in a medium supplemented with 4.4 pM BA and 87.6 sucrose (initiation medium). Presence of NAA in the medium caused callus growth. Auxin did not promote culture initiation
Fig. 2. Shoot growth of A. cordata subcultured from initiation medium (MS + 4.4#M BA + 87.6 mM sucrose) to multiplication medium (MS + 1.1 #M BA + 333 mM glucose) (4 weeks after incubation in multiplication medium - 6th subculture).
 and shoot multiplication [11, 12] in other Alnus species either. Transfer of cultures from initiation medium to a medium with reduced
BA (1.1 #M) and supplemented with glucose (333 mM) instead of sucrose (