Plant regeneration of Fraxinus angustifolia by in vitro shoot organogenesis

Scientia Horticulturae 87 (2001) 291±301

Plant regeneration of Fraxinus angustifolia by in vitro shoot organogenesis G. Tonona,*, M. Capuanab, A. Di Marcob a

Dipartimento di Colture Arboree, UniversitaÁ di Bologna, Via F. Re 6, 40126 Bologna, Italy b Istituto Miglioramento Genetico delle Piante ForestalõÂ, CNR, Via Vannucci 13, 50134 Firenze, Italy Accepted 6 May 2000

Abstract The in vitro regeneration of Fraxinus angustifolia through bud/shoot organogenesis on embryonic explants was investigated. Embryo axes and cotyledons from mature seeds underwent adventitious regeneration after exposure to growth regulators, the embryo axes showing a greater regenerative potential than cotyledons. While the MS medium proved effective for the induction and initiation phases, it was inadequate for bud development, and was replaced by DKW medium. A constant supply of benzyladenine (BA) appeared essential for all phases. The gelling agents used had an important role in morphogenesis: Duchefa Gelrite positively affected the number of new buds per explant. The rooting of developed shoots was easily achieved on auxin-free WPM medium and most of the rooted plantlets ``hardened'' after a period of acclimation in a mist greenhouse. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Ash; Micropropagation; Shoot organogenesis; Rooting

1. Introduction Fraxinus angustifolia, a characteristic component of Mediterranean forest vegetation (Bernetti, 1995), is a fast-growing tree that produces wood of good

Abbreviations: BA, 6-benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; 2-iP, 2-isopentenyladenine; NAA, 1-naphthaleneacetic acid; TDZ, thidiazuron * Corresponding author. Tel.: ‡39-051-209-1490; fax: ‡39-051-209-1500. E-mail address: [email protected] (G. Tonon). 0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 7 8 - 3

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quality and is suitable for both lowland and sub-mountain areas. That it is much valued as an ornamental also (Lopez, 1984) makes this species useful both in urban forestry and for ornamental purposes, particularly in the southern countries of Europe. Improvement of the species through breeding programs is thus an important goal, and in clonal strategies the integrated use of in vitro biotechnology can help to shorten the time generally required by conventional breeding methods (Cheliak and Rogers, 1990). Although the clonal propagation of ash by cuttings is frequently unsuccessful (Dirr and Heuser, 1987), there are some reports of in vitro regeneration of Fraxinus species. For example, both shoot organogenesis and somatic embryogenesis in white ash (F. americana L.) were achieved using cut mature seeds as primary explants (Preece et al., 1987a, 1989; Bates et al., 1992, 1993; Bates and Preece, 1995); and in common ash (F. excelsior), adventitious shoot formation from different explants was attained (Hammatt, 1996) while micropropagation has been achieved from juvenile (Chalupa, 1990; Leforestier et al., 1990; Hammatt and Ridout, 1992) and mature (Hammatt, 1994) plants. Yet there are no reports of adventitious regeneration via organogenesis or somatic embryogenesis for F. angustifolia, and only one of micropropagation, in which plant material from both in vitro germinating seedlings and 10-year-old trees was used (Perez-Parron et al., 1994). The present study investigated in vitro regeneration of F. angustifolia via bud/shoot organogenesis from embryonic explants for the production of viable rooted plants. 2. Materials and methods 2.1. Plant material Mature embryos extracted from non-strati®ed seeds collected from local trees during autumn 1996, and stored in the dark at 428C were used as explants. The samaras were imbibed with distilled water for 48 h, the pericarps removed and the seeds sterilized in 70% ethanol solution for 3 min, followed by 20 min soaking in 1.8% sodium hypochlorite solution and three rinses in sterile distilled water. The embryos were extracted by cutting the seed along the borders and dissected as per the following trial protocols. 2.2. Culture conditions All nutrient media contained 20 g lÿ1 sucrose; the pH was adjusted to 5.8 with KOH or HCl after dissolving gelling agents and before autoclaving (20 min at 1208C). Eight explants per replicate were initially placed into 60 mm diameter plastic Petri dishes, each containing 10 ml solidi®ed medium; 25150 mm

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culture tubes containing 12 ml medium were then used for root induction. All cultures were incubated at 2328C with a 16-h photoperiod under 80 W ``coolwhite'' ¯uorescent lamps to provide 2.5 W mÿ2 photosynthetically active radiation. 2.3. Regeneration and rooting procedures In the ®rst trial, the in¯uence of a 55 factorial combination of BA and 2,4-D, both at 0, 0.44, 2.2, 4.4 and 8.8 mM was tested on embryo axes and cotyledons for the induction of regeneration. Half-strength MS (Murashige and Skoog, 1962) medium for macroelements and full-strength organics and microelements, solidi®ed with 3 g lÿ1 Duchefa Gelrite, was used. After 30 days of culture, the number of regenerating explants (with at least one bud) was recorded. Four replicates with a total of 32 explants per treatment were carried out. In the second trial, the development of regenerated buds into shoots was studied starting from a new culture cycle of primary explants cultured on the regeneration medium that in the previous experiment had given the best results (MS, halfstrength for macroelements, supplemented with BA 4.4 mM and 2,4-D 0.44 mM). After 30 days on the regeneration medium, explants with buds were transferred to shoot development media consisting of half-strength (macroelements) MS or DKW (Driver and Kuniyuki, 1984) formulations solidi®ed with Duchefa Gelrite (3 g lÿ1), and supplemented with BA 0, 0.44, 2.2, or 4.4 mM. Fourteen explants per treatment were individually placed in glass culture tubes and, after 30 days, the number of developing shoots per culture with at least one node was recorded. In the third trial, the regenerative potential of different embryo explants with or without the root and apical meristem portions (Fig. 1) was tested using the same nutrient medium and the best hormone combination of the ®rst trial (BA 4.4 mM

Fig. 1. Diagram of a F. angustifolia embryo, indicating explants tested in the third experiment.

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and 2,4-D 0.4 mM). Four replicates with a total of 32 explants per treatment were tested. In the fourth trial, carried out using the same medium, the in¯uence of different gelling agents on the regeneration process was tested: Duchefa Gelrite (3 g lÿ1), Serva Gelrite (3 g lÿ1), and Difco Bacto Agar (7 g lÿ1) were compared by culturing embryo axes (devoid of root and apical meristems) and cotyledons. Eight replicates for a total of 64 explants per treatment were tested. In the third and fourth experiments, the number of regenerating explants and the number of visible buds per explant were recorded after 30 days of culture. A ®nal experiment on rooting was carried out in which regenerated shoots at least 1 cm in height were excised and placed on to half-strength DKW or fullstrength WPM (Lloyd and McCown, 1980) media solidi®ed with Duchefa Gelrite with or without 5.0 mM NAA. At least 10 baby food jars (containing 25 ml medium) with a total of 40 explants were used per treatment. After 40 days, the number of rooted shoots and the number and length of roots of each rooted shoot were recorded. Plantlets taller than 30 mm were set in a bench ®lled with a peat± sand±perlite substrate and received natural daylight in a greenhouse. The programmed mist system was initially set for 90% relative humidity and gradually reduced to about 60% over 4 weeks. All experiments, except the rooting test, were replicated at least twice. 2.4. Statistical analysis All data, except rooting percentages, were subjected to analysis of variance using the Sigmastat Program; the difference in the mean values among different factors being evaluated with the Student±Newman±Keuls method. In order to normalize the data, all percentage values were converted to radians using an arcsin (Y)1/2 transformation (Gomez and Gomez, 1976). Data on the ®rst two levels of BA (0 and 0.44 mM) tested in the ®rst experiment with the embryo axes and all the data for cotyledons were excluded from statistical analysis for a surplus of 0%. Data on rooting percentages were analyzed by a w2 test. 3. Results Organogenetic phenomena appeared on the explants after 10±13 days' culture (Fig. 2). In the ®rst trial, the embryo axes expressed a higher morphogenic ability than cotyledons, which regenerated occasionally. A direct organogenetic process appeared in both types of explant, mostly on wounded tissues in contact with the medium. No callus production associated with regeneration was observed. Analysis of variance showed a signi®cant interaction between the two growth regulators tested (BA and 2,4-D) for the induction of regeneration (Table 1).

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Fig. 2. Organogenesis on explants of F. angustifolia: adventitious bud/shoot formation on an embryo axis (A) and a portion of cotyledon (B) (barsˆ1 mm).

Organogenesis occurred only when the BA concentration was at least 2.2 mM. By contrast, 2.4-D at the highest level tested (8.8 mM) inhibited regeneration and its presence in the medium at low concentrations was not essential in inducing the regenerative process. Thus, at every level of BA concentration tested the regenerative response with BA alone was never statistically lower than that observed by combining BA and 2,4-D. In the second experiment, the DKW medium was more effective than MS as a secondary medium at all BA concentrations (Table 2). The number of fully

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Table 1 Effect of growth regulators on the adventitious regeneration (percentage of regenerating explants) observed on embryo axes and cotyledons after 30 days of culturea BA (mM)b

2.4-D (mM)c

Mean

0.00

0.44

2.20

4.40

8.80

0.00 0.00 a50.00b a75.00cd a53.12b

0.00 0.00 a43.75b b90.62d a37.50b

0.00 0.00 a12.50a b40.62b c65.62b

0.00 0.00 a0.00a b50.00bc b40.62b

0.00 0.00 a3.12a a0.00a a0.00a

59.37c

57.29c

39.58b

30.20b

1.04a

Cotyledons 0.00 0.44 2.20 4.40 8.80

0.00 0.00 0.00 3.12 3.12

0.00 0.00 6.25 9.37 9.37

0.00 0.00 3.12 0.00 3.12

0.00 0.00 0.00 6.25 0.00

0.00 0.00 0.00 0.00 0.00

Mean

2.08

8.36

2.08

2.08

0.00

Embryo axes 0.00 0.44 2.20 4.40 8.80 Meand

0.00 0.00 a21.87 c51.15 b39.37

0.00 0.00 1.87 3.75 3.12

a

Main effect: for BA P<0.001, for 2,4-D P<0.001; interaction: for BA2,4-D P<0.001. Values from the ®rst two levels of BA applied to embryonic axes and all values referring to cotyledons were excluded from the statistical analysis. c Values on the rows and values on the columns respectively followed or preceded by at least one equal letter are not statistically different (P<0.05), according to the Student±Newman±Keuls method. Letters at the values' right refer to the 2,4-D treatment; letters at the left refer to the BA treatment. d The means were calculated on the last three levels of BA. b

Table 2 Effect of secondary medium and BA concentration on the development of elongated shoots (n) carrying at least one nodea BA (mM)b

Medium DKW MS Mean a

Mean

0.00

0.44

2.20

4.40

b1.21a a0.07a

b1.71a a0.29a

b3.57b a0.71b

b3.74b a1.07b

0.64a

1.00a

2.14b

2.36b

a2.54 b0.54

Main effect: media, P<0.001; BA, P<0.001; interaction: mediaBA, Pˆ0.054. Values on the rows followed by different letters and values on the columns preceded by different letters are statistically different at P<0.05 according to the Student±Newman±Keuls method. Letters at the values' right refer to the BA treatment; letters at the left refer to the medium. b

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Table 3 Organogenetic capacity (percent of regenerating explants and mean number of new buds per explant) of different embryo portions after 30 days of culture Portion

Regenerating explants (%)a

Buds per explant (n)

A B C D E F G H

9.37d 87.50a 65.62bc 0.0 50.00c 6.25d 75.00abc 77.38ab

1.67b 5.43a 2.33b 2.63b 1.50b 3.13b 3.56b

a Values on the columns followed by at least one equal letter are not statistically different (P<0.05) according to the Student±Newman±Keuls method.

developed shoots rose along with increasing BA concentration, in both DKW and MS media. No shoots developed from cotyledons. The third experiment con®rmed the limited regenerative capacity of cotyledons and their presence in the explants resulted in a strong inhibition of organogenetic potential, as evidenced by comparing results of portions F, D and C (Fig. 1; Table 3). While the portion nearest to the root meristem (portion A) showed low regeneration ability; the embryo axes devoid of root and apical meristems (portion B) exhibited the highest regeneration capacity in terms of number of new adventitious buds per explant; no statistically signi®cant differences were observed between the latter portion and portions G and H in terms of percentage of regenerating explants (Table 3). Even in the fourth experiment no regeneration was observed on cotyledons. While the kind of gelling agent did not in¯uence the percentage of regenerating explants, Duchefa Gelrite had a signi®cant positive effect on the number of new buds per explant (Table 4). The NAA treatment did not appear to be essential for rooting (Fig. 3), since similar rooting was achieved with WPM devoid of auxin (Table 5). Nor was Table 4 Effect of gelling factors on the percentage of regenerating explants and on the mean number of new buds per explant, observed on embryo axes devoid of the apical and root meristems Gelling factors

Regenerating explants (%)a

Buds per explant (n)a

Duchefa Gelrite Serva Gelrite Difco Bacto Agar

78.12a 65.62a 81.25a

5.64a 3.10b 3.00b

a Values on the column followed by different letters are statistically different at P<0.05 according to the Student±Newman±Keul method.

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Fig. 3. In vitro rooted plantlet of F. angustifolia (barˆ1 cm).

Table 5 Effect of nutritive solution and auxin supply on rooting percentage, and mean number and length of roots per rooted shoot, after 40 days of culture Medium

NAA (mM)

Rooting (%)a

Roots per shoot (n)b

Root length (cm)b

DKW DKW WPM WPM

0.0 5.0 0.0 5.0

25.00b 63.64a 65.00a 60.00a

1.45c 3.11b 1.96c 4.17a

2.71a 1.78b 2.96a 1.61b

a

Values on columns followed by different letters are statistically different at P<0.05 according to the w2 test. b Values on columns followed by different letters are statistically different at P<0.05 according to the Student±Newman±Keuls method.

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hormone-free DKW medium suitable for rooting. Although NAA positively affected the number of roots per shoot, it adversely affected root length. While the roots that developed after the NAA treatment had no secondary roots, shoots rooted on the hormone-free media always exhibited rooting systems with secondary roots (data not reported). Eighty percent of rooted plantlets survived the acclimation in greenhouse and are now growing in a nursery shade area. 4. Discussion and conclusions The embryo axes and cotyledons excised from mature seeds of F. angustifolia underwent adventitious shoot regeneration after exposure to growth regulators, the former showing higher organogenetic capacities than the latter. This ®nding is similar to that observed by Preece et al. (1989) in white ash (F. americana L.), where somatic embryogenesis and bud organogenesis were only achieved on cotyledons that were still connected to the zygotic embryo axes. These ®ndings suggested that the germinating seeds enhance the regenerative processes by supplying certain substances (e.g. hormones) lacking in the cotyledons. In our study, the wounds appeared to be an important inducing factor for organogenetic processes. It may be that the cut-induced hormone stress plays a role in adventitious bud induction since the best organogenetic response at least in terms of new bud number per explant, was achieved on embryo axes after a double-cut of their apical and root meristems. Similar results were reported by Hammatt (1996), who cultured the distal and proximal portions of transversely cut cotyledons. Morphogenetic processes like rooting, somatic embryogenesis and bud organogenesis are often described as complex phenomena characterized by different phases, each with speci®c nutritional requirements (Thorpe, 1988; Gaspar et al., 1992). Indeed, in the present study, MS solution proved to be effective for the induction and initiation phases but inadequate for bud development. The DKW medium, characterized by a high Ca2‡ ion concentration, was more suitable than MS for bud development; the inef®ciency of MS as secondary medium was also reported by Bates et al. (1992) on white ash. On the other hand, in terms of the growth regulator requirements, a constant supply of BA appeared essential for all regeneration phases. This fact stands in contrast to what has been found with F. americana, where neither BA nor 2-iP was able to induce the formation of adventitious structures, which appeared only when the explants were cultured on media containing thidiazuron (Bates et al., 1992). Similarly, F. excelsior on TDZ performed better in culture than BA, evincing more frequent regeneration and fewer culture deaths (Hammatt, 1996). The important role played by gelling agents in the morphogenetic processes (Debergh, 1983; Ripetti et al., 1994) was con®rmed in the present study: during

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the induction and initiation phases, it affected the number of new buds per explant but not the percentage of regenerating explants. The rooting of developed shoots was easy even on auxin-free WPM medium, as also found by Silveira and Cottignies (1993) in F. excelsior. The suitability of WPM as rooting medium for Fraxinus spp. has been con®rmed in other studies too (Preece et al., 1987b; Perez-Parron et al., 1994). Overall, the reported data indicate that bud organogenesis from embryo axes of F. angustifolia is a viable approach to regeneration in this species. The fact that the different embryonic tissues tested expressed a high (embryo axis) and low (cotyledon) regenerative potential suggests that further investigations at the biochemical and molecular levels are needed to gain a better understanding of the morphogenetic processes induced from embryonic organs. References Bates, S., Preece, J.E., 1995. Somatic embryogenesis in white ash (Fraxinus americana L.). In: Jain, S., Gupta, P., Newton, R. (Eds.), Somatic Embryogenesis in Woody Plants, Vol. 2. Kluwer Academic Publishers, Dordrecht, pp. 311±325. Bates, S., Preece, J.E., Navarrete, N.E., Van Sambeek, J.W., Gaffney, J.R., 1992. Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.). Plant Cell Tiss. Org. Cult. 31, 21±29. Bates, S.A., Preece, J.E., Yopp, J.H., 1993. Secondary somatic embryogenesis and plantlet conversion of white ash. HortScience 28, 405. Bernetti, G., 1995. Selvicoltura Speciale. UTET, Torino. Chalupa, V., 1990. Micropropagation of hornbeam (Carpinus betulus L.) and ash (Fraxinus excelsior L.). Biol. Plant. 32, 332±338. Cheliak, W.M., Rogers, D.L., 1990. Integrating biotechnology into tree improvement programs. Can. J. For. Res. 20, 452±463. Debergh, P., 1983. Effect of agar brand and concentration on the tissue culture medium. Physiol. Plant 59, 270±276. Dirr, M.A., Heuser, C.W., 1987. The Reference Manual of Woody Plant Propagation from Seed to Tissue Culture. Varsity Press, Athens, GA. Driver, J.A., Kuniyuki, A.H., 1984. In vitro propagation of paradox walnut root-stock. HortScience 19, 507±509. Gaspar, Th., Kevers, C., Hausman, J.F., Berthon, J.Y., Ripetti, V., 1992. Practical uses of peroxidase activity as a predictive marker of rooting performance of micropropagated shoots. Agronomie 12, 757±765. Gomez, K.A., Gomez, A.A., 1976. Statistical Procedures for Agricultural Research, 2nd Edition. Wiley, New York. Hammatt, N., 1994. Shoot initiation in the lea¯et axils of compound leaves of micropropagated shoots of juvenile and mature common ash (Fraxinus excelsior L.). J. Exp. Bot. 45, 871±875. Hammatt, N., 1996. Fraxinus excelsior L. (Common Ash). In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry, Trees IV. Springer, Berlin, pp. 173±193. Hammatt, N., Ridout, M.S., 1992. Micropropagation of common ash (Fraxinus excelsior L.). Plant Cell Tiss. Org. Cult. 31, 67±74.

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