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Les Laboratoires Rhizotec Inc., St-Jean Chrysostome, C.P. 797, Quebec, Canada G6Z 2L9;. (present address: ... Dawson 1979; Dawson 1983). To evaluate the ...
New Forests 3 : 225-230 (1987) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands Research n o t e

Commercial micropropagation of five Alnus species

P. P I ~ R I N E T & F . M . T R E M B L A Y Les Laboratoires Rhizotec Inc., St-Jean Chrysostome, C.P. 797, Quebec, Canada G6Z 2L9; (present address: Petawawa National Forestry Institute, Canadian Forestry Service, Chalk River, Ontario, Canada KOJ IJO) Received 10 December 1986; accepted 27 April 1987

Key words: Actinorhiza, alder, tissue culture, Frankia Alder micropropagation techniques were adapted for large scale production. Clonal micropropagation can be accomplished on a commercial basis if: - the level of iron in the multiplication medium is increased to 70 mg/L of Sequestrene 330Fe, and the NH4NO3 concentration is decreased to 500 mg/L depending on the species, the shoots are subcultured once on regular MS medium prior to the rooting treatment, - the rooted plantlets are gradually transferred from controlled mist conditions to normal greenhouses or shelters.

Application.

Clonal micropropagation was demonstrated as feasible on a commercial basis for several clones of five Alnus species. Approximately 60,000 ready-to-root individual shoots were multiplied in vitro on modified MS medium supplemented with 2.5-5 ttM BAP. A total of 15,500 shoots from different clones were rooted in vitro on half strength MS medium including 1-5 ttM IBA. They were transferred under mist conditions within a growth chamber illuminated with high pressure sodium lamps. Those conditions gave 99-100% plantlet survival after four weeks. Plantlets were then inoculated with selected Frankia sp. strains. These nodulated alder plants are under field evaluation at the Petawawa National Forestry Institute, Canadian Forestry Service in Chalk River, Ontario.

Abstract.

Introduction

A c t i n o r h i z a l plants like alders, which have the ability to fix a t m o s p h e r i c n i t r o g e n t h r o u g h symbiotic association with the a c t i n o m y c e t e Frankia, are v a l u a b l e species i n forestry for b i o m a s s p r o d u c t i o n , soil i m p r o v e m e n t , a n d g r o w t h i m p r o v e m e n t o f associate vegetation ( T a r r a n t & T r a p p e 1971; G o r d o n & D a w s o n 1979; D a w s o n 1983). T o evaluate the potential o f selected clonal alder m a t e r i a l u n d e r field c o n d i t i o n s , 13 clones f r o m five A l n u s spp. were m i c r o p r o p a g a t e d c o m m e r c i a l l y . M i c r o p r o p a g a t i o n t e c h n i q u e s have been previously described for seven alder species ( G a r t o n et al. 1981; Read et al. 1982; P6rinet & L a l o n d e 1983; T r e m b l a y & L a l o n d e 1984; T r e m b l a y et al.

226 1984). These techniques had to be adapted to meet the needs of commercial production, where continuous and repeated subculturing often results in mineral deficiency, morphological malformations, or necrosis of the cultures.

Materials and methods

Micropropagation

The following species and clones were used: - A . glutinosa (L.) Gaertn., clones AG-I., AG-2 (P6rinet & Lalonde 1983), AG-3, AG-4, AG-8 (Tremblay & Lalonde 1984) - A . incana (L.) Moench., clones AI-1, AI-2 (id.) - A . j a p o n i c a (Thunb.) Steud., clones A J-6, A J-7 (id.) A . rubra Bong., clones ARb-8, ARb-10 (id.) - A . crispa (Ait.) Pursh., clones AC-15 (id.), non-nodulating AC-4 (Nod-) (Tremblay et al. 1984). These clones were originally initiated from seedlings or two year-old plants. The techniques and culture media were according to Tremblay and Lalonde (1984) but with NaFeEDTA replaced by Sequestrene 330Fe at 70 mg/L. Cultures were incubated in a culture room (Fig. 1) at a constant 25+ I°C under Vita-Lite fluorescent tubes (Duro-Test Electric Ltd., Ont.) providing 45-100 /zEm--2s-1 with a 16/8 h day/night photoperiod. Shoots were multiplied with 2.5 ~M benzylaminopurine (BAP) except A . crispa clones which were produced on 5 #M BAP. A . incana, A . j a p o n i c a , and A . rubra clones were multiplied on a modified MS medium where NH4NO~ was lowered to 500 mg/L. Shoots were subcultured once every four weeks on the multiplication medium. Prior to the rooting treatment, they were subcultured once on regular MS multiplication medium. Rooting media (half strength MS salts) included: - 3% glucose for clones AI-1, AI-2, AJ-6, AJ-7, ARb-8, ARb-10 - 3% sucrose for clones AG-1, AG-2, AG-3, AG-4, AG-8 - 1.5% glucose for clones AC-4, AC-15 In rooting media, indolebutyric acid (IBA) concentration was: - 1 #M for clones AG-1, AG-2, AG-3, AG-4, AG-8, AI-1, AI-2 - 2.5 ttM for clones AC-4, A J-6, A J-7, ARb-8, ARb-10 - 5 #M for clone AC-15 From the 60,000 shoots multiplied in vitro, 1,000 to 2,500 shoots per clone were rooted. Rooted plantlets were washed in tap water and transplanted into IPL containers (IPL Ltd, St-Damien, Qu6.) filled with a peat:vermiculite substrate (Redi-Earth, Terra-Lite, F. Hyde Co., Montr6al, Qu6.). They were -

227

Fig. 1. Culture room for in vitro multiplication and rooting; Fig. 2.Multiplication stage of clone AG-1 in 1.5 L Mason jar. Bar = 1 cm; Fig. 3. Micropropagated plantlets of clone AJ-7 in IPL container five weeks after soil transfer and showing a fast and uniform development. Bar = 3 cm; Fig. 4. Acclimated micropropagated alder plants in growth room 4-5 weeks after transfer.

maintained

under

mist c o n d i t i o n s

for t w o

weeks in g r o w t h

chambers

i l l u m i n a t e d with high pressure s o d i u m lamps ( L u m i p o n i c Inc., B o i s b r i a n d , Qu6.) at 25 +_ 1 ° C with 16/8 h d a y / n i g h t p h o t o p e r i o d . O v e r the first three-

228 week-period, illuminance was regularly increased from 100 to 175/xEm--2s -1 and finally 300/zEm--2s -1, concomitant with decreasing humidity.

Frankia inoculation

One to two weeks after transfer to the soil, plants of all clones, except AC-4, were inoculated as previously described (P6rinet et al. 1985) with the Frankia strains ACN14a, AGNlg, TN18gcb, and ARgN22d (Lalonde et al. 1981; Normand & Lalonde 1982) in nitrogen-free Crone's solution.

Results and discussion

High rates of multiplication (Fig. 2), similar to those reported in the literature, were obtained for all 13 clones from five species. This enabled the production of more than 60,000 shoots within 5-6 months, including the stock build-up period from an initial low number of cultures. Sequestrene 330Fe at 70 mg/L corrected the apical chlorosis occasionally observed with NaFeEDTA during multiplication. Repeated transfers of A. incana, A. japonica and A. rubra on regular MS medium brought a higher frequency of shoot malformations and basal necrosis, together with a decrease of shoot production. These problems were solved by lowering the NH4NO3 concentration in the MS medium. It was also found that low nitrogen medium (31.3 mM of total N) decreased shoot length and diameter compared to MS medium (60 mM of total N). Therefore, multiplication was made on low nitrogen medium, followed by a final subculture on regular MS medium. This procedure resulted in the maximum number of shoots with improved characteristics for rooting. For 11 out 13 clones, rooting percentages, determined with 1,000 to 2,500 shoots per clone, varied between 92-100070 (Table 1). This successful commercial production confirmed results already published. By contrast, under the conditions described in this paper, the clones AC-4 and AC-15 rooted at 62070 and 88070, respectively (Table 1), which is lower than previously described (Tremblay et al; 1984; Tremblay & Lalonde 1984) These dissimilar results might be related to different light sources and different illuminance levels during the rooting treatment. For example, we used Vita-lite tubes at 45-100 /zEm--2s -~ instead of the Cool-White:Gro-Lux WS combination previously used at 125/~Em--2s -~. Rooting of clone AC-4 was slower than other clones, taking 4-5 weeks instead of 2-3 weeks. Even after that period, half of the plantlets had only necrotic root primordia. Survival percentages after four weeks in the growth room (Figs. 3, 4) were

229 Table 1. Rooting and survival percentages and total number of plants produced per clone.

Clone AC-4 AC- 15 AG-1 AG-2 AG-3 AG-4 AG-8 AI-I AI-2 A J-6 A J-7 ARb-8 ARb-10

Rooting (%o)

Survival (%)

Total number of plants

62 88 97 96 92 95 100 97 96 97 100 95 96

76 98 99 100 100 99 100 99 99 99 100 99 99

1,500 225 1,100 200 1,150 200 1,950 1,500 2,450 1,125 1,900 1,100 1,100

99-100%, except in the case of clone AC-4, where 24°7o of the plantlets failed to develop (Table 1). Poor root formation during the rooting treatment might explain the lower survival observed with this clone. A total of 15,500 plants were produced, with 1,100 to 2,500 plants per clone, except for clones AG-2 and AG-4. Of these latter clones only 200 plantlets out of 1,000 were needed for transfer to soil (Table 1). The inoculation treatments resulted in 100% nodulation after 2-3 weeks.

Conclusion It is feasible to produce, on a commercial basis, five alder species including the non-nodulating A. crispa clone AC-4 (Nod-). Modification of the nitrogen content in the multiplication medium was necessary for A. incana, A. japonica and A. rubra, so as to adjust the described techniques to a commercial level. The techniques used to produce 15,500 plants could have been easily extended to the available 60,000 shoots or more. Potential production on an annual basis for all species tested was evaluated at over 1 million plants per clone. Field evaluations are currently being conducted that will provide information on genotypic stability and true-to-type uniformity of these micropropagated plants.

Acknowledgments We thank Claire Filion and Alain Marchand for assistance with the plant material.

230 This

work

was

partly

supported

by th e

Canadian

Forestry

Service

(DSS:52SS.KH415-4-0664), by a P I L P p r o j e c t (CA910-4-0024/B12) with the N R C o f C a n a d a , a n d by an i n v e s t m e n t l o a n ( V I D 6132-2-9070-03-02D) f r o m the M A P A Q , Qu6bec.

References Dawson, J.O. 1983. Dinitrogen fixation in forest ecosystems. Can. J. Microbiol. 29: 979-992. Garton, S., Hosier M.A., Read, P.E., and Farnham, R.S. 1981. In vitro propagation of Alnus glutinosa Gaertn. HortSci. 16:758-759. Gordon, J.C., and Dawson, J.O. 1979. Potential uses of nitrogen-fixing trees and shrubs in commercial forestry. Bot. Gaz. 140 (Suppl.):S88-S90. Lalonde, M., Calvert, H.E., and Pine, S. 1981. Isolation and use of Frankia strains in actinorhizae formation, p. 296-299 In Gibson, A.H. and Newton, W.E., (eds) Current perspectives in nitrogen fixation. Austral. Acad. Sci. Canberra. Normand, P., and Lalonde, M. 1982. Evaluation of Frankia strains isolated from provenances of two Alnus species. Can. J. Microbiol. 28:1133-1142. P6rinet, P , Brouillette, J.G., Fortin, J.A., and Lalonde, M. 1985. Large scale inoculation of actinorhizal plants with Frankia. Plant and Soil 87:175-183. P6rinet, P., and Lalonde, M. 1983. In vitro propagation and nodulation of the actinorhizal host plant Alnus glutinosa (1.) Gaertn. Plant Sci. Lett. 29:9-17. Read, P.E., Garton, S., Louis, K., and Zimmerman, E.S. 1982. In vitro propagation of species for bioenergy plantation, p. 757-758 In Fujiwara, A. (ed.) Plant Tissue Culture 1982. Tokyo. Tarrant, R.F., and Trappe, J.M. 1971. The role of Alnus in improving the forest environment. Plant and Soil (Special Vol.): 335-348. Tremblay, F.M., and Lalonde, M. 1984. Requirements for in vitro propagation of seven nitrogenfixing Alnus species. Plant Cell Tissue Organ Culture 3:189-199. Tremblay, F.M., Nesme, X., and Lalonde, M. 1984. Selection and micropropagation of nodulating and non-nodulating clones ofAlnus crispa (Ait.) Pursh. Plant and Soil 78:171-179.