Plant Science Letters, 29 (1983) 9--17 9 Elsevier Scientific Publishers Ireland Ltd.

IN VITRO PROPAGATION AND NODULATION OF THE ACTINORHIZAL HOST PLANT A L N U S G L U T I N O S A (L.) GAERTN.

P. PI~RINET* and M. LALONDE

D~pt. Ecologie et Pddologie, FacultJ de Foresterie et G~od~sie, Universitd Laval, Qudbec G1K 7P4 (Canada)

(Received March 30th, 1982) (Revision received July 13th, 1982) (Accepted August 26th, 1982)

SUMMARY

Multiple shoots were formed from seedling cultures of Alnus glutinosa (L.) Gaertn. on modified Murashige and Skoog medium supplemented with 0.5--5 pM of benzyladenine (BAP). Formation of both adventitious and axillary shoots ensured a high rate of multiplication. Rooting of the shoots occurred after transfer of the individual shoots to media either containing indolebutyric acid (IBA) or lacking growth regulator. Direct rooting of the shoots was also possible under non,sterile conditions. Plantlets of Alnus glutinosa were inoculated with a pure culture of Frankia ACN1AG for initia- tion of nitrogen-fixing actinorhizae. Two different clones of A. glutinosa were propagated in vitro, nodulated by Franl~ia ACN1AG and grown in a nitrogen-free substrate. Rooting, nodulation and growth recovery was 100% for the two clones.

Key words: Micropropagation -- Tissue culture - -Alnus glutinosa --Frankia -- Nitrogen fixation -- Actinorhizae

INTRODUCTION

Nitrogen-fixing actinorhizal plants are largely utilized in such forestry practices as reforestation, land reclamation and biomass production. Of these plants, alder species are of particular interest because of their growth potential and their genetic variability [ 1 ]. At present, little is known of the genetics of the actinorhizal species utilized in forestry and most of them are

Abbreviations: BAP, benzyladenine; IBA, indolebutyric acid; 2iP, N6-(A2-isopentenyl) - adenine; NAA, a-naphthaleneacetic acid. *Present address: Montreal Botanical Garden, 4101 E. Sherbrooke, Montreal, Quebec, Canada H1X 2B2.

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0304-4211/83/0000---0000/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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wild genotypes. Alnus glutinosa is an introduced species extensively planted in the United States. Breeding with native species should improve the cold hardiness of A. glutinosa and increase its utilization.

Genetic amelioration of alder involves selection of particular traits and recombination of the desired traits by means of hybridization [1]. Already identified Superior genotypes and new ones that will be selected will need to be propagated through cloning [1,2]. Vegetative propagation of Alnus glutinosa through cuttings is possible using standard horticultural techniques [3,4]. But conventional propagation for large-scale multiplication requires nursery facilities like mist beds and lath houses and the establishment of clonal orchard to maintain sufficient stock-plants to pro'vide the needed cuttings. In vitro propagation, through shoot or bud cultures, ensures a faster multiplication of a new selected genotype giving in a year around production a large number of genetically identical and disease-free plants [5,6l.

Manipulation of the actinorhizal symbiosis between Alnus and the N2- fixing actinomycetal endophyte, Frankia, involves genetic studies of both the host plant and the Frankia in order to maximize the biological nitrogen fixation. Since the interactions of the genotypes of plant and endophyte may represent a significant component of the phenotypic variation [ 7 ], a genetic amelioration program should consider the symbiotic state of the plant with the Frankia.

Plant tissue culture is now recognized as a valuable tool for genetic amelio- ration of trees [8]. Torrey [9], Vasil et al. [10] and Giles and Vasil [11] emphasized the importance of plant tissue culture technology, in particular protoplast and cell culture, for the in vitro study of symbiotic nitrogen fixation. The successful application of these new approaches in cell culture will depend on the regeneration of plants from cell and tissue cultures [ 10]. Sommer and Brown [2] made an exhaustive review of the applications of tissue culture for forest tree improvement and indicated that some species of genera of the following dicotyledonous trees could now be propagated by in vitro techniques: Populus, Eucalyptus, Betula, Liquidambar, Acacia and Ulmus. Additional species were reported recently in the following genera: Castanea [ 12], Tectona [ 13] and Santalium [ 14]. Alnus species have been cultivated in vitro for callus initiation [15,16].

Root nodules of A. glutinosa have also been cultivated in order to grow the endophyte in host tissue [17]. We now report the in vitro propagation of A. glutinosa and the successful nodulation of the plantlets by a pure culture of Frankia ACN1 AG .

MATERIALS AND METHODS

Explant Seeds of A. glutinosa (L.) Gaertn. (lot. no. 778930, 1977, F.R.G., supplied

by the Canadian Forestry Service, Petawawa, Ontario) were soaked for 24 h

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at 4°C, then disinfected in a 30% solution of H202 with a drop of Tween 20 for 20 min and rinsed with 1 1 of sterile distilled water. The treated seeds were placed in Petri dishes containing water agar (0.5%) and glucose (0.5%) in order to detect microbial contaminants. The germinated seeds were then transferred to Murashige and Skoog medium containing 5 - t 0 pM of BAP, including or lacking 0.1 ~M of a-naphthaleneacetic acid (NAA). After 6 weeks on the MS medium, the epicotyl part of the seedling, either with or wi thout the cotyledons, was transferred on MS medium containing 5 uM of BAP.

Media The basal culture medium consisted of mineral salts according to

Murashige and Skoog [18] , 3% sucrose, vitamins as in B5 medium [19] and 0.7% Bacto Difco Agar. Either casein hydrolysate (0.05%) or glycine (2 mg/1) was added to the medium. The pH was adjusted to 5.8 with NaOH or HC1, before autoclaving the medium. BAP, N6-(zX2-isopentenyl)adenine (2iP), NAA or IBA were added to the basal medium individually or in combination, at different concentrations. Unless otherwise stated, the multiplication medium consisted of the basal medium with 1 ~M of BAP. The culture medium was distributed in glass tubes (20 X 150 mm) or in Erlenmeyer flasks of 250 ml and autoclaved at 1210C for 15 min at 15 lb/in 2. Tubes were closed by plastic caps and flasks with foam plugs. The water used for the media was obtained from a reverse osmosis system (Culligan International Co., Nor thbrook, IL, U.S.A.). For the rooting of the shoots, the salts were at half-concentration of the basal medium.

Cultural conditions The shoot cultures were incubated in a growth chamber at a constant

temperature of 26°C or 24°C, 16-h photoper iod and at 70% or more relative humidity. During the multiplication step, the light intensity was 2500 lux. The light source consisted of a mixture of fluorescent (Cool-White, Sylvania) and 60V¢ incandescent lamps. For the rooting of the shoots, a range of 2--10 klux was used with a thermoperiod of 24°C and 20°C at night.

Transfer to artificial substrate After 14 or 21 days on rooting medium, the rooted shoots were first

washed in tap water and then transferred to s tyroblock planting containers No. 8 (Mansonville Plastics Ltd, Longueuil, Canada) containing a nitrogen- free artificial substrate Turface (Plant Products Co., Ontario, Canada) under mist conditions in a greenhouse. Plants were fertilized every 2 weeks with nitrogen-free Crone's solution [21] .

Frankia inoculum A pure culture of Frankia, strain ACN1 A~ grown in Qmod medium of

Lalonde et al. [20] , was used as inoculum to induce nodulation of A.

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glutinosa plantlets. They were inoculated 1 - 2 days after their transfer to the greenhouse.

Acetylene reduction assay Excised nodules from the roots of in vitro propagated A. glutinosa were

used for the acetylene reduction assay. The root nodules (syn.: actinorhizae) were incubated for 1 h in a 10% (v/v) acetylene in air. Samples of 0.2 ml of the gaseous phase were analysed for C2H4 in a Hewlett~Packard gas chromatograph according to a modified procedure of Koch and Evans [ 22 ].

Nitrogen content analysis Leaves and stems from 3--4-month-old plants were dried for 60 h at

70°C, ground and subjected to Kjeldahl determination following the proce- dure of Coles and Parks [23] .

RESULTS

Initiation After 5 weeks on the basal medium containing 5--10 ~M of BAP, the

epicotyl and the roo t of the alder seedling had not elongated and the epi- cotyl had formed a rosette of leaves. Shoots developed from the rosette and/or from the cotyledonary nodes. The tissue bearing the elongating shoots was subcultured and new shoots and buds formed at the base of the shoots. No buds differentiated from the cotyledons or the hypocotyl . With time, callus formed on the cotyledons. In one case, an adventitious bud was formed at the margin of a leaf in one of the rosettes.

No at tempts were made to determine the percentage of alder seedlings capable of forming multiple shoots when cultivated in presence of BAP, or in what manner the auxin/cytokinin ratio could affect their formation. Initially we cultivated 5 clones f rom 5 different seedlings. For the regenera- t ion studies, however, only two of them were used and they responded in the same manner to the different steps of the propagation procedure.

Multiplication When the alder initial explant was subcultured on the multiplication

medium, the shoots elongated and axiUary buds developed at the base of the shoots. Clusters of shoots and buds were formed rapidly and could be separated for subculture. A good multiplication rate was achieved by the continuous development of axillary buds and by the formation of numerous adventitious buds in the nodal regions (Fig. 1). These adventitious buds differentiated directly on the basal part of the shoots wi thout previous formation of a callus.

Different concentrations of BAP and 2iP, with or wi thout IBA, were tried in the multiplication medium to determine the optimal concentration of cytokinin. The multiplication rate, i.e. the number of elongating shoots

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2

Fig. 1. Multiple shoots formation on multiplication medium with 1 uM of BAP after 3 weeks of culture. Bar = 10 ram.

Fig. 2. Initiation of roots on an individual shoot ofA. glutinosa with IBA at 1 ~M, after 14 days. Bar = 10 ram.

p e r e x p l a n t , a n d t h e g r o w t h o f t h e s e s h o o t s w e r e a l w a y s h i g h e r on a m e d i u m

c o n t a i n i n g B A P t h a n w i t h 2 iF ( T a b l e I) . B A P a t 0 .5 , 1 a n d 5 / ~ M i n d u c e d

t h e f o r m a t i o n a n d d e v e l o p m e n t o f n u m e r o u s s h o o t s . A t h i g h e r c o n c e n t r a -

t i o n s (10 a n d 25 p M ) b r o w n ca l lus was f o r m e d on t h e p a r t s o f t h e e x p l a n t s

in c o n t a c t w i t h t h e m e d i u m a n d t h e d e v e l o p m e n t o f t h e c u l t u r e s was l o w e r .

T h e a d d i t i o n o f I B A a t 1 /~M in t h e m u l t i p l i c a t i o n m e d i u m c o n t a i n i n g

TABLE I

EFFECTS OF BAP, 2iP AND IBA ON ORGAN FORMATION OF A. GLUTINOSA SHOOT CULTURES AFTER 21 DAYS ON THE MULTIPLICATION MEDIUM

A minimum of 10 explants per treatment were used and were subcultured 3 times on the same media.

BAP (~M) 2iP (~M) IBA (/~M) Organ formation

Callus Shoot Root

0.5 0 -- + + + -- I 0 ~ + + + -- 5 0 -- + + + -- 5 1 + + + + +

I0 0 + ++ -- 25 0 + ++ --

5 0 -- + + 5 1 + + +

I0 0 -- + + 50 0 -- + + 80 0 ~ + +

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5 pM of BAP or 2iP did not increase the multiplication rate but induced the format ion of callus and roots on shoots and leaves.

The multiplication rate ranged from 1 to 15 elongating shoots per explant after 21 days of culture in presence of 1 pM of BAP. This rate was a function of the size and the development of the explant. Buds and bud primordia were no t included in that number, only the elongating shoots of 5 mm and more. Fif ty shoots, ranging from 0.5 cm to 5 cm, could be obtained from the same flask containing 3--6 explants after 21 days on the multiplication medium. The cultures were maintained for more than one year with subcul- turing every 3 weeks on the multiplication medium.

R o o t init iation Inciividual shoots were excised from 3-week-old cultures on multiplication

medium and placed in tubes containing the rooting medium. The length of shoots ranged from 1 cm to 3 cm, a few of them were up to 5 cm. Different concentrations of IBA were tried in the rooting medium: 0, 1, 2.5 and 5 pM. For these four concentrations, the first roots appeared after 7 days and the rooting was 100% after 14--28 days (Fig. 2). The number of roots per shoot increased with the raise in concentrat ion of IBA. Higher concentrations of IBA (2.5 pM and 5 pM) caused formation of callus, though Very limited in

L I!i

ALNUS GLUTINOS,'~

8"7"81

PHOTO : 13' 11-81 ,.

Fig. 3.4-month-old nodulated plants of A. glutinosa grown in a nitrogen-free substrate. B a r - 5 cm.

Fig. 4. Rooting system with numerous effective nodules of a plant after 4 months of growth in styroblock container. N, nodules; Sh, base of the shoot. Bar = 5 mm.

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TABLE II

SURVIVAL, NODULATION, HEIGHT, ACETYLENE REDUCTION AND NITROGEN CONTENT OF TWO CLONES OF IN VITRO PROPAGATED A. GLUTINOSA

Clone no.

AG-I AG-2

Survival (%) 100 100 Nodulation (%) 100 100 Height (cm) 3-month-old plants 23.5 a 22.3 b Acetylene reduction nmol C:H,/mg nod. dry wt./h 25--36 30--57 Nitrogen content (%)

Leaves 3 3 Stems 1.2 1.2

aMean of 8 plants. bMean of 6 plants.

size. With auxin in the rooting medium, 90--100% of the shoots were rooted after 14 days and 100% after 21 days. For the control, wi thout IBA, the rooting was 100% after 21--28 days.

Transfer to artificial substrate and nodulation by Frankia Alnus glutinosa plantlets were transferred to s tyroblock containers with

a nitrogen-free substrate, and maintained under intermittent mist conditions for 10 days in a greenhouse. Afterwards, they were transferred to a growth chamber. The survival of the plantlets was 100% and all of them were nodulated following inoculation with the Frankia ACN1AG {Fig. 4). The effective nodules were able to sustain normal growth of the alder plantlets fertilized with the nitrogen-free Crone's solution (Fig. 3). Table II summa- rizes the height, the rate of acetylene reduction and the nitrogen content of the two in vitro propagated clones of A. glutinosa.

As the rooting of the shoots was maximum without auxin in the rooting medium, unrooted shoots of A. glutinosa from the multiplication stage were directly transferred to Turface under intermittent mist and inoculated with Frankia ACN1AG . Both the rooting and the recovery of growth were 100%.

DISCUSSION AND CONCLUSION

Alnus glutinosa was easily propagated by in vitro techniques from seedling material, BAP at 0.5--5/~M induced the formation of numerous axillary and adventitious buds, ensuring a high regeneration rate. The formation of adventitious buds emphasizes the organogenetic potential of that material. That allows us to speculate that meristem culture from older plants of A. glutinosa could manifest a good organogenetic response.

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The rooting of the shoots was 100% after 14-21 days of culture. IBA at 1 ~M gave a more uniform and a faster rooting of the shoots than the control and without formation of callus. Ten days under the mist conditions were sufficient to allow complete hardening of the plantlets. The plantlets started to grow immediately after the transfer to Turface. The effective nodulation of the plantlets by the Frankia ACN1 AG ensured the adequate nitrogen nutrition of the plants. Direct transfer of the unrooted shoots from the cultures to Turface under mist conditions was possible and this should be taken into consideration for large scale production.

It is now possible to obtain numerous clones of A. glutinosa. These represent an inestimable experimental tool for the genetic study of the Alnus-Franleia symbiosis. The high organogenetic potential of the seedling material should facilitate the propagation of mature trees of superior geno- types. Successful results recently obtained in clonal propagation of mature trees [13,24] and in somatic embryogenesis in cultures of sweetgum [25] and birch [26] are encouraging outcomes of the application of tissue culture to forest genetics.

Note Added in Proof Following the revision of this paper, Garton et al. (Hort Science, 16 (1981)

758) reported the micropropagation of A. glu tinosa in similar conditions.

ACKNOWLEDGEMENTS

We are indebted to Mr. G. Pr~gent for the nitrogen content determination and to Ms. S. Berch for reviewing the manuscript. This paper was made possible by a grant (No. 7192) from NSERC of Canada to M.L.

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