Plant Cell, Tissue and Organ Culture 15:85-89(1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands

Short communication

In vitro propagation of mature trees of Alnus incana (L.) Moench.

P. PI~RINET 1, G. VALLI~E 2 & F.M. T R E M B L A Y 1 Les Laboratoires Rhizotec Inc., C.P. 797, St-Jean-Chrysostome, Quebec G6Z 2L9, Canada; (present address: ; Petawawa National Forestry Institute, Canadian Forestry Service, Chalk River, Ontario KOJ 1JO, Canada; :Ministbre de l'Energie et des Ressources Quebec, Direction de la Recherche et du D~veloppement, 2700 Einstein, Ste-Foy, Quebec G1P 3W8, Canada

Key words: Alnus incana, alder, micropropagation, mature trees

Abstract. Mature trees of European grey alder (Alnus incana) were micropropagated on a modified MS medium containing 2.5/~M BA, 6.2mM (500mgl -~) NH4NO 3 and 1.5% glu- cose. Prior to in vitro culture, mature scions were multiplied through grafting and cutting techniques. Shoot tips from cuttings were established in vitro. After six months of culture, shoots were rooted either in vitro or in vivo and plantlets were transferred to greenhouse conditions.

Introduction

Alder species have received particular at tention for soil improvement and biomass production because of their nitrogen-fixing symbiotic association with the actinomycete Frankia [1]. The European grey alder (A. incana ssp. incana) is a tree that can reach 25 m in height, and is easily distinguished from the shrubby American speckled alder (A. incana spp. rugosa) which rarely exceeds 5-7 m.

Alder micropropagation, including that of A. incana has been reported for seedlings and young trees [2, 4, 6, 7, 9] and has been recently reviewed [8]. However, the feasibility o f in vitro propagation from mature alder trees has yet to be demonstrated. We now report in vitro propagation of mature trees (20- to 25-years-old) of A. incana through shoot-tip culture.

Materials and methods

Plant material

Mature A. incana trees: AI-39 (45-years-old), AI-41, AI-44, and AI-46 (all 25-years-old), and AI-45 (20-years-old), grow as a naturalized populat ion at

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Pointe Platon, Qurbec. They are putative offspring from trees introduced to Canada at the beginning of the century and have been selected naturally for cold resistance.

Dormant twigs bearing pistillate and staminate inflorescences were collec- ted from several trees in February 1985. These mature scions were grafted onto 3- to 4-year-old A. glutinosa plants using approach grafting with the scion base submerged in water. Following vegetative growth of the graft scions in a greenhouse, one-leaf internode cuttings were rooted and esta- blished as mother plants for in vitro culture.

In vitro culture

The cuttings (5-33 cuttings per clone) produced buds that were available for in vitro propagation. Growing shoot tips were excised from these cuttings on 30 September 1985. They were disinfected and explanted according to Tremblay & Lalonde [7]. The basal culture medium consisted of Murashige & Skoog minerals and vitamins without KI, 70 mg 1- ~ Sequestrene 330Fe as the iron source, 1.5% (w/v) glucose, and 0.7% (w/v) Bacto-agar (Difco). The pH was adjusted to 5.5 before autoclaving. Basal medium was used unless otherwise stated. Benzyladenine (BA) at 2.5/~M was added to the basal medium for the establishment and multiplication steps.

Explants were established and kept in 20 x 150 mm culture tubes for 13 weeks. Axenic explants were first transferred as small shoot clumps after five weeks and, thereafter, at monthly intervals. At the third subculture, after 13 weeks, cultures were transferred for multiplication to 1.5-liter Mason jars containing 200 ml of basal medium but with NH4NO3 lowered to 6.2 mM (500 mg 1 -l ) [5].

All cultures were tested before the third subculture for microbial contami- nants on QmodB medium [3].

Cultures were incubated in a growth room at a temperature of 25 °C and a 16/8 h day/night photoperiod under 45/~mol m -2 s-~ provided by Vita-Lite fluorescent lamps (Duro-Test Electric Ltd., Ont.). The light intensity was increased to 90/~mol m -2 s -~ after 7-10 days of rooting treatment.

In vitro rooting and soil transfer

In vitro-produced shoots, 1.5 to 2.5cm high, were rooted in Mason jars containing half-strength basal medium with 1 #M indolebutyric acid (IBA) and glucose raised to 3% (w/v) [7]. After 18-19 days on the rooting medium, plantlets were transferred into IPL containers No. 45-110 (IPL Ltd, St-

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Damien, Qu&) filled with a soilless mix containing peat and vermiculite 1:1 and 2000 ppm of soluble 20-20-20 commercial fertilizer. Containers were placed in the 'bouturath6que' (see below) for one week under a light inten- sity of 60pmol m -2 s -~ before being transferred to greenhouses.

In vivo rooting

Some in vitro-produced shoots were rooted under non-sterile conditions in the 'bouturath6que', a device designed by the Service de la Recherche, Minist6re de l'Energie et des Ressources Qu6bec, for rooting broad-leaved and conifer cuttings. The 'bouturath6que' consists of numerous confined enclosures (244cm long x 40cm high x 60 or 122cm wide) made of acrylic and plexiglass, and assembled as multistoried shelves.

For in vivo rooting, 135 microcuttings of the clone AI-45, 1.5 to 2.5 cm high and similar to the shoots used for in vitro rooting, were transferred directly into IPL containers filled with the soilless mix described earlier. The microcuttings were handled as regular cuttings. Containers were placed in the 'bouturath6que' for four weeks under a light intensity of 35 pmol m -2 s- with a 16/8 h day/night photoperiod.

Results

Culture establishment

The apical and axillary buds from five clones of A. incana, AI-39, AI-41, AI-44, AI-45, and AI-46 (14 to 63 buds per clone), were excised from the growing shoot tips and explanted in vitro. After nine weeks of growth, the total contamination rate varied from 21% to 43%.

Both apical and axillary buds developed shoots in vitro without phenolic exudation into the medium. However, after four weeks, several apical buds were overgrown by callus produced from stipules and leaf petioles. Subse- quent browning of the stipule tissues coincided with an inhibition of further development of the buds; these cultures were eliminated.

Successful in vitro establishment varied among clones. Clones AI-41 and AI-45 responded well to in vitro initiation, with 36% of the explanted buds being axenic and established as shoot cultures after 13 weeks, compared to 6% for clone AI-46 and 0% for clones AI-39 and AI-44. Despite an initial outgrowth of the buds for clone AI-39, none survived beyond the second subculture. The sole growing culture of clone AI-44 was contaminated. After elimination of contaminated and/or non-developing explants, three clones out of five were multiplied.

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Multiplication

Multiplication of the clones AI-41, AI-45, and AI-46 was achieved on a modified MS medium containing 6.2 mM NH4NO3. After three months of multiplication, 200 to 650 shoots ( > 1.5 cm) were produced per clone. This variation in shoot number was related to the variable number of initiated cultures rather than to different multiplication rates. Multiplication rates were estimated to be three- to four-fold every month of multiplication.

Rooting

Individual shoots of AI-41 (49 shoots), AI-45 (161 shoots), and AI-46 (31 shoots) were excised and rooted under in vitro conditions. The first roots appeared after seven days in the rooting medium. Rooting was 84% for AI-41, 99% for AI-45 and 90% for AI-46, after 14--19 days, with two to six roots per rooted shoot.

Of the 135 microcuttings of clone AI-45 rooted under in vivo conditions, 91 (67.4%) were rooted after six weeks compared to 99% when rooted in vitro.

Both types of plantlet survived after being transferred to greenhouses.

Discussion

Three out of five mature trees of A. incana, 20- to 22-years-old, were successfully propagated in vitro following a modified technique previously developed for two-year-old plants [7]. However, explants of two Alnus trees failed to develop further in spite of an initial outgrowth of the buds.

Mature material was micropropagated using a medium with low nitrogen concentration (31.3mM of nitrogen instead of 60mM in regular MS medium), as already used by Ptrinet & Tremblay [5] for commercial micro- propagation of several clones of A. incana, A. japonica, and A. rubra.

The rooting ability, under in vitro conditions, of shoots initiated from mature trees was comparable to results reported for cultures initiated from juvenile material [7]. A lower rooting percentage was obtained with clone AI-45 under in vivo conditions compared to in vitro conditions. However, this difference was more related to the handling of microcuttings in the bouturathtque than to their rooting ability per se. The in-vitro-produced shoots, treated as conventional cuttings, were found to be more sensitive to desiccation than regular cuttings.

Clones of mature plants were established in vitro using explants taken from rooted cuttings obtained from the original grafted scions. The neces-

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sity for such a step was not determined. The cuttings used as mo the r plants

still showed some plagiotropism, while the mic rop ropaga ted plants were

fully o r tho t rop ic and had juvenile leaves morphologica l ly similar to those in

seedlings. The mic rop ropaga ted clonal plants f rom three A. incana mature trees are now under field evaluat ion.

Acknowledgements

This work was pe r fo rmed at Les Labora to i res Rhizotec Inc. and was par t ly

suppor ted by the Service de la Recherche, Minist6re de l 'Energie et des Ressources Qu6bec. We are grateful to Claire Filion for technical assistance. Field selection o f the trees and their p ropaga t ion by graft ing and cuttings,

and plantlet handl ing in the boutura th6que , were pe r fo rmed by the Service de la Recherche, Minist6re de l 'Energie et des Ressources Qu6bec.

References

1. Dawson JO (1983) Dinitrogen fixation in forest ecosystems. Can J Microbiol 29:979-992 2. Garton S, Hosier MA, Read PE, Farnham RS (1981) In vitro propagation of Alnus

glutinosa Gaertn. HortScience 16:758-759 3. Lalonde M, Calvert HE (1979) Production of Frankia hyphae and spores as an infective

inoculant for Alnus species. In: Gordon JC, Wheeler CT, Perry DA (Eds) Symbiotic Nitrogen Fixation in the Management of Temperate Forests, pp 95-110. Oregon State University, Corvallis

4. P6rinet P, Lalonde M (1983) In vitro propagation and nodulation of the actinorhizal host plant Alnus glutinosa (L.) Gaertn. Plant Sci Lett 29:9-17

5. P6rinet P, Tremblay FM (1987) Commercial micropropagation of five Alnus species. New For 3:225-230

6. Read PE, Garton S, Louis K, Zimmerman ES (1982) In vitro propagation of species for bioenergy plantations. In: Fujiwara A (Ed) Plant Tissue Culture 1982, Proc 5th Int Cong Plant Tissue Cell Cult, Tokyo, pp 757-758

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9. Tremblay FM, Nesme X, Lalonde M (1984) Selection and micropropagation ofnodulating and non-nodulating clones of Alnus crispa (Ait.) Pursh. Plant and Soil 78:171-179