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 (87.6mM) improved growth, producing satisfactory shoot multiplication and elongation (Table 1, Fig. 2). Reduction of BA concentration to 1.1 #M, incubation in the dark, application of GA3, or use of activated charcoal did not improve shoot multiplication in the presence of 87.6 mM sucrose. Shoot multiplication was greater in the presence of 333 mM glucose than 87.6 mM sucrose. Shoot elongation was greater in the presence of glucose (in the light) or sucrose (in the dark or in the presence of activated charcoal) than cultures grown in a medium containing 87.6 mM sucrose and incubated in the light. The overall results suggested that initial culture of explants in 'initiation medium' (MS medium containing 4.4#M BA and 87.6 mM sucrose) and their subsequent transfer (after 7 days) to shoot 'multiplication medium' (MS medium containing 1.1 #M BA and 333 mM glucose) was the most
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suitable treatment for shoot production. This could be repeated until enough shoots were produced (two-stage shoot production). Once preference of A. cordata for glucose at culture initiation stage was establish- ed, media containing 1.1 or 4.4#M BA + 333mM glucose for culture initiation and shoot multiplication (one-stage shoot production) were tried and results comparable to two-stage shoot production were achieved (av- erage shoot number and shoot length of 2.3 and 9.2mm, respectively). Regenerated shoots could be cut to smaller pieces containing 2-3 axillary buds and be subcultured for further shoot multiplication. After 2-3 subcul- tures, an average shoot multiplication of four to fivefold could be obtained in this way every 4-5 weeks.
Requirements for different sugars between and within some Alnus species (i.e.A. crispa, A. incana, A. japonica, A. rubra, A. sinuata andA. viridis) has been reported [14, 15]. Tremblay & Lalonde  used a two-stage procedure (i.e., culture initiation, shoot multiplication) for shoot production of Alnus species listed above. The major difference between the two stages was either an increased sucrose concentration or replacement of sucrose by glucose in the shoot multiplication stage. Read et al.  and Garton et al.  used a one-stage shoot production procedure (i.e., the same medium for culture initiation and shoot multiplication containing 87.6 mM sucrose). Read et al.  used sucrose for micropropagation of A. glutinosa, A. crispa and A. rubra, but a comparison between the effect of different sugars on shoot multiplication was not made. Tremblay & Lalonde  reported that .4. crispa and A. rubra preferred glucose, while A. glutinosa preferred sucrose for shoot multiplication. The present study indicates that although initially glucose is preferred by .4. cordata (Table 1), at a later stage (after 6 regenera- tion passages) sucrose could fulfil the sugar requirements of .4. cordata (Table 2).
Table 2. The effect of sucrose and glucose on in vitro shoot production of A. cordata (in their 7th passage) after 4 weeks on 1.1/~M BAJ
Sucrose Glucose LSD (87.6mM) (5%)
166.5 mM 333 mM
Shoot number 2.59 2.73 2.28 1.26 Shoot length (mm) 8.85 10.20 8.72 4.88 Shoot tip necrosis/ 0.09 0.36 0.00 0.19 shoot number
A. cordata shoot cultures were grown for 6 passages in vitro prior to this experiment; each passage consisted of culture of explants in MS + 4.4 #M BA + 87.6 mM sucrose for 7 days and then transfer of cultures to MS + 1.1 #M BA + 333mM glucose for a further 20-25 days.
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There were no significant differences in shoot multiplication and elonga- tion in the media containing 1.1 pM BA supplemented with 87.6 mM sucrose or 166.5-333 mM glucose after cultures had been grown for 6 passages in vitro, suggesting an in vitro conditioning had taken place. However, there was a significant increase in shoot tip necrosis among shoots produced in the medium containing 166.5mM compared to 333mM glucose (Table 2). Prolonged culture under optimum growth conditions can induce a partial or a complete rejuvenation in some plants, e.g. Robinia pseudoacacia (black locust) , Cyphomandra betacea (tamarillo)  and Vitis vinifera . Such an in vitro conditioning usually promotes shoot growth and rooting, reflect- ing a change in the physiological status of the explants. Replacement of sucrose by glucose enhanced the in vitro rooting of the 'difficult-to-root' clones of Alnus species . It is possible that sugars play a role in in vitro conditioning. This preference of Alnus species for different sugars, reported in literature [15, 16] and in this study, may not only be related to individual preference (species or clones) but also be due to the physiological status of the cultured explants.
Fig. 3. In vitro root growth on an A. cordata shoot cultured in MS medium (1/2 x macronu- trients) supplemented with 4.9 #M IBA and transferred to hormone-free medium 10 days after culture initiation (2 weeks after culture).
Shoot elongation was improved by dark incubation, and addition of activated charcoal in media containing 87.6mM sucrose. Application of GA 3 did not improve elongation (Table 1).
In MS medium (1/2 macronutrients), in vitro rooting was best achieved with 1.2-4.9#M IBA and 87.6/~M sucrose. Rooting occurred within 2-3 weeks (Table 3, Fig. 3). Sucrose was a better source of sugar, particularly for production of root number, than glucose for in vitro rooting (except at 4.9/~M IBA). Transfer of shoots from media containing 333 mM glucose and 4.9/~M IBA to hormone-free media after 10 days improved rooting. Cytological observation indicated the presence of some root initials after 7 days. Prolonged exposure to auxin can reduce rooting by reduction of root development, and by excessive callus growth [1, 3]. Root initiation and root growth and development are two separate processes and root
Fig. 4. In vivo rooting of an A. cordata shoot in potting mix after exposure of shoots at the end of in vitro shoot multiplication stage to liquid MS medium supplemented with 0.123 mM IBA for 24 h (3 weeks after transfer to potting mix).
Table 4. Effect of IBA concentrations on rooting of A. cordata shoots regenerated in vitro)
0 0.025 0.123 0.615 3.076
Root 4.00 4.50 4.48 5.00 7.2 number 2 Rooting 81 80 93 95 98 (%)
t MS medium containing IBA (0-3.076 mM) was poured into culture jars covering 10 mm of the base of the shoots (at the end of shoot multiplication in vitro) for 24 h; these shoots were cut to 15-20 mm pieces that were used as mini-cuttings and placed in potting mix to root. Rooting was recorded after 3 weeks in potting mix. 2 LSD (5%) for root number: 1.8.
development conditions can be inhibitory to the initiation of further root primordia [7, 9]. Incubation of shoots in the dark during the initial six days, in the presence of glucose, did not improve rooting. Many shoots which had not produced roots during the in vitro treatments subsequently rooted in potting mix (Table 3).
In vitro produced shoots (10-20 mm) rooted well in potting mix (Fig. 4). Addition of a root-promoting liquid medium (MS medium supplemented with 3.076 mM IBA) to the culture jars for 24 h at the end of shoot multi- plication, and prior to taking of mini-cuttings, improved rooting significant- ly (Table 4). This method of in vivo rooting has a significant economic advantage over in vitro rooting as work is carried out under non-aseptic conditions and away from culture room. Application of liquid media con- taining rooting hormone was reported , but at lower concentrations (9.8-19.6 pM IBA), in aseptic conditions, and over a period of 7 to 14 days.
This study has established procedures for micropropagation of A. cordata plants originating from mature mother plants.
The following method is recommended for micropropagation of mature A. cordata: - Establish stem cuttings from field-grown plants in a glasshouse. - Culture actively growing sterilized axillary bud explants in MS medium containing 1.1-4.4/tM BA and 333 mM glucose. After 4-5 weeks, cut elon- gated shoots to pieces containing 2-3 axillary buds and culture them again in the same medium till enough shoots are regenerated. - Root regenerated shoots as mini-cuttings (15-20mm) in MS medium (1.2 macronutrients) containing 4.9pM IBA; alternatively, place re- generated shoot clusters in liquid MS medium supplemented with 3.076 mM IBA covering 10 mm of the base of the shoots for 24 h and then cut them to mini-cuttings, as above, and put them in potting mix to root.
The author gratefully acknowledges receipt of a New Zealand National Research Advisory Council (NRAC) post-doctoral fellowship and supply of material from Soil Conservation Centre, Ministry of Works and Develop- ment, Aokautere.
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