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Role of basal shoot darkening and exogenous putrescine treatments on in vitro rooting and on endogenous polyamine changes in difficult-to-root woody species
Scientia Horticuiturae, 53 ( 1993 ) 63-72 Elsc,..,ier Science Publishers B.V., Amsterdam
63
Role of basal shoot darkening and exogenous putrescine treatments on in vitro rooting and on endogenous polyamine changes in difficult-toroot woody species Eddo Rugini, Antonella Jacoboni and Maria Luppino Istituto di Ortofloroarboricoltura, Universitit della Tt,~cia, Facolti~ di Agraria, Via S. Camillo de Lellis, Ol lO0 Viterbo, Italy (Accepted 6 July 1992)
ABSTRACT Rugini, E., Jacoboni, A. and Luppino, M., 1993. Role of basal shoot darkening and exogenous putrescine treatments on in vitro rooting and on endogenous polyamine changes in difficult-to-root woody species. Scientia Hortic., 53: 63-72. The effect of in vitro putrescine treatment on several difficult-to-root fruit tree species was studied in order to improve rooting efficiency. Putrescine was added to a double liquid layer at the shoot proliferation phase and during the rooting phase. Shoots were rooted in either a i 6-h photoperiod or a 16-h photoperiod with shoot bases in the dark (basal darkening). Putrescine, at I mM, in a double liquid layer, increased the number of pear shoots with expanded leaf lamina but did not increase the rooting efficiency of these shoots. Putrescine increased the rooting of apple (M9 rootstock, clone P3 ) and the basal explants of olive, rooted in 16-h photoperiod; decreased it in walnut but did not affect chestnut, almond, jojoba and apricot rooting. Basal shoot darkening was essential for root formation in chestnut, walnut and almond. The endogenous content of perchloric acid (PCA)-soluble free polyamines (PA) was determined a~ the end of the proliferation phase in olive, chestnut and walnut. The shoots proliferated under the (~-h photoperiod contained a lesser concentration of free spermidine and spermine compared with mo~c kept in the dark for 10 days before being excised for rooting. High levels of spermine were detected in walnut shoots. Total free polyamine content was relatively low in olive, at an intermediate le,Jei in chestnut and high in walnut. The content seems to be inversely correlated with the response to exogenous putrescine treatments. In addition, PCA-soluble free and FCA-soluble bound polyamines in olive shoots, both basal and apical, either in a 16-h photoperiod or in basal darkening, showed an increase at Day 2 of rooting and then decreased in the following days. In 16-h photoperiods basal explants of olive, which under these conditions require applied exogenous putrescine for a strong rooting response, accumulated all three PA as bound forms at Day 2. However, those which rooted with basal shoot darkening did not respond to exogenous putrescine or accumulate PA. Keywords: almond; apricot; chestnut; Chinese pear; jojoba; olive; pear; walnut.
Correspondence to: E. Rugini, Instituto di Ortofloroarboricoltura, Universit/l della Tuscia, Facolt/~ di Agraria, Via S. Camillo de Lellis, 01100 Viterbo, Italy.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4238/93/$06.00
64
E. RUGINI ET AL.
Abbreviations: agar = agar purified (Sigma); BA = benzyladenine; DFMA = difluoromethylarginine; D F M O = difluoromethylornithine; GA3 = gibbereUic acid; 1BA = indolebutyric acid; NAA = alpha-naphthaleneacetic acid; PA = polyamine; PCA = perchloric acid.
INTRODUCTION
Rooting results from a complex of biochemical and physiological processes affected by several factors. Rooting in vitro is affected by genetics, juvenility, growth regulators, phenolic compounds, shoot quality of previous subcultures, peroxidase activity, photoperiod, light intensity and quality, etc. (Nemeth, 1986). In particular, it is well known that darkness during the first week of the rooting phase is often essential in stimulating rooting in several woody species (Druart et al., 1982; Sriskandarajah et al., 1982; Rugini and Verma, 1983), although, with dark pretreatment, survival of plantlets usually decreases when they are transferred to the soil (Von Arnold and Erikson, 1984). Darkening only the basal part ofolive and almond explants during the rooting phase, by painting the outside of the jar black and covering the medium surface with black polycarbonate granules, can replace the initial period of darkness required for an increase in rooting efficiency (Rugini et al., 1988 ). Among the several factors that can affect root induction, the endogenous polyamines (PA) may play an important role (Sankhla and Upadhyarya, 1990). The inhibition of PA synthesis by specific inhibitors such as diflouromethylarginine (DFMA) and difluoromethylornithin¢ (DFMO) inhibits rooting. This effect can be partially reversed by exogenous PA treatment (Biondi et al., 1990). The effect of exogenous PA treatment on rooting stimulation is not well understood and appears to be dependent on the plant species and the physiological state of the shoots before excising the explants or the plant before collecting the cuttings (Rugini et al., 1992). Exogenous PA treatment of olive, when combined with auxins, appears to promote earlier and higher rooting percentage ofboth in vitro explants and cuttings (Rugini and Wang, 1986). Therefore this work pursued two objectives: ( 1 ) to test the effect of exogenous putrescine on other fruit tree species; (2) to analyze the polyamine variation in olive during the first days of rooting, and in olive, chestnut and walnut shoots at the end of their proliferation phase. These species were chosen for their different responses to putrescine treatment, as putrescine positively affects olive rooting, but has no effect on chestnut rooting and decreases rooting in walnut. MATERIALS AND METHODS
Some difficult-to-root cultivars of the following species were used in this study: almond, apple, apricot, chestnut, Chinese pear rootstock, jojoba, olive,
IN VITRO ROOTING IN WOODY SPECIES
65
pear and walnut. With pear and Chinese pear rootstock, putrescine was added filter-sterile in the double liquid layer, which was incorporated on the solidified medium at the end of the culture cycle. Putrescine was added to the rooting medium for the other species. The following species, environmental conditions and media were adopted in this study: Pear (Pyrus communis L.), cultivar 'Kaiser', maintained on MS (Murashige and Skoog, 1962) medium plus 2.25 ~tM BA, 0.5/iM IBA, 3% sucrose and 0.7% agar with a double liquid layer and putrescine; Chinese pear rootstock (Pyrus calleryana), maintained on woody plant medium (WPM) salt medium (Lloyd and McCown, 1981 ) plus 0.66/zM BA, 0.08/zM NAA, 0.2 /~M GA3, 3% sucrose and 0.7% agar at pH 6, with a double liquid layer and with or without putrescine; walnut (Juglans regia L.), unknown ecotype, maintained on DKW (Driver and Kuniyuki, 1984) mineral salts medium plus 4.5/~M BA, 0.05/~M IBA, 3% sucrose and 0.7% agar, apple (Malus pumila), M9 rootstock, clone P3, maintained on MS basal salt medium plus 3.5 /iM BA, 3% sucrose and 0.7% agar at pH 5.8; chestnut (Castanea sativa Miller), maintained on DKW medium plus l ~tM BA, 3% sucrose and 0.7% agar; jojoba (Simmondsia chinensis (Link.) Schneider), maintained on MS medium plus 3.5/iM BA, 0.08/~M NAA, 3% sucrose and 0.7% agar; apricot (Prunus armeniaca L.), cultivar 'Bebeco', maintained on SH (Schenk and Hildebrandt, 1972) macroelements, MS microelements plus 3.5/~M BA, 3% sucrose and 0.7% agar; almond (Prunus dulcis Batsch), cultivar 'Ferraduel', maintained on DKW mineral salts plus 3.5/tM BA, 0.08/~M NAA, 3% sucrose and 0.7% agar, with 3-week subcultures; olive (Olea europaea L. ), cult:,var 'Moraiolo', maintained on olive (OM) medium (Rugini and Fedeli, 1990) plus 5/~M of zeatin. Olive, chestnut and walnut shoots were placed in the dark l 0 days prior to excising the microcuttings in order to verify how the dark was able to affect endogenous PA levels before the rooting phase. All shoots were derived from buds collected from l 0-15-year-old mature bearing plants, except for jojoba and walnut, which were derived from l-yearold and 5-year-old seedlings, respectively. All cultures were started at least 2 years earlier and maintained in 500 ml jars with 100 ml of its own proliferation medium and with 20 explants per jar. They were then placed in a growth room at 23°C +_ l °C, under fluorescent white cool light at 40/~mol m -2 s -~ with a 16-h photoperiod. The double liquid layer medium for pear and Chinese pear rootstock contained the mineral salts of MS plus 3% sucrose, l/zM BA and l/zM IBA at pH 5.5 with or without l mM putrescine. At the end of the culture cycle, 30 ml of the liquid medium was added to the agar solidified one as described by Maene and Debergh ( 1985 ), and left for 28 days; the shoot explants were then excised from cultures for the rooting test. Rooting was carried out in 120 mm × 16 mm test tubes with 5 ml Bourgin and Nitsch (1967) medium with macro elements reduced to half, plus 2% sucrose, with or without 5/~M NAA and 0.7% agar. One apical explant, 2-3
66
E. RUGINI ET AL.
cm long, was placed in each tube with a total of 60 explants for each treatment. The tubes were then placed in a growth room with the same environmental conditions used for the proliferation phase. The rooting experiment of olive, given its strong apical dominance, differed from the others because the entire shoot was divided into two parts (apical and basal) of equal length (two to three nodes) and kept separate. Apical explants were kept separated from the basal ones because, in our experience, putrescine only rarely affects rooting in olive apical explants in terms of early root promotion and increase of rooting percentage. The treatments, for all species, were as follows: ( 1) control ( 16-h photoperiod); (2) control with 1 mM of putrescine; (3) control with basal darkening; (4) control with basal darkening and with 1 mM of putrescine. The filter-sterilized putrescine was added, after autoclaving, before medium solidification. The black sterile polycarbonate granules (small cylinder of 2 mm in diameter and 2.5 mm high, from Montedison) were placed, before implanting the explants, on the surface of solidified medium in a layer of about 4 mm, in order to m~ke the medium dark. The outside of the tubes were painted black up to the medium level as previously described (Rugini et al., 1988). An additional sample of olive explants were prepared for endogenous polyamine determination. Rooting percentage was observed on the Day 28 and data were subjected to the analysis of variance, by subdividing the test tubes in four blocks of 15 tubes each. Polyamine extraction and analysis. - Two kinds of plant material were ana-
lyzed for PA content: ( 1) whole explants of olive at 0, 2, 5, and 8 days from the beginning of the rooting experiments; (2) olive, chestnut and walnut shoots, by discarding the basal part in contact with the medium, previously placed for 10 days in the dark, in comparison with the shoots grown in a 16h photoperiod. Each sample was subdivided into two repetitions of 0.5 g each and frozen in 2.5 ml of 5% cold perchloric acid (PCA). The samples were homogenized with Ultraturrax at a progressively increasing speed for about l min and centrifuged at 26000 × g for 10 min. The pellet was resuspended in 2.5 ml PCA and centrifuged again. A sample of 2.5 ml of the combined two supernatants and the pellet were separately hydrolyzed in 6 N HCI for 18 h at 110°C in vials. The hydrolysates were evaporated under vacuum and resuspended in the original volume of PCA. Aliquots of the PCA supernatant (free PA), of the same hydrolyzed supernatant (conjugated PA ) and of the hydrolyzed pellet (bound PA) were dansylated according to Palavan and Galston (1982) and Tonigiani et al. (1987) and separated by thin layer chromatography in chloroform:triethylamine (5:1), then detected with a spectrofluorimeter.
IN VITROROOTINGIN WOODYSPECIES
67
RESULTS
In all species the explants did not root without auxin addition both those treated or untreated with putrescine (data not shown ). Basal shoot darkening and putrescine affected rooting according to the species, type of explants and the endogenous PA content at the end of the proliferation phase. P e a r a n d C h i n e s e pear. - Most of the pear shoots, proliferated on a double liquid layer medium with putrescine, showed fully expanded leaves com~ pared with those without putrescine (Table 1 ). However, the microcuttings excised from these shoots, when placed on the rooting medium without putrescine, rooted in less percentage compared with those collected from a double liquid layer without putrescine. The basal darkening method seemed to affect, negatively, rooting of shoots proliferated without putrescine. However, in Chinese pear, putrescine added to a double liquid layer, did not affect leaf expansion or the rooting of the microcuttings, while basal darkening increased the rooting percentage of the explants (Table 1 ). Putrescine increased rooting percentage both in the control explants and in those with basal darkening. However, the basal darkening alone did not affect rooting (Table 2 ).
Apple. -
W a l n u t , c h e s t n u t a n d a l m o n d . - Rooting occurred only with the basal darkening method and putrescine had no effect on rooting except in walnut where rooting was significantly decreased by this treatment (Table 2 ).
The basal darkening treatment increased rooting over the control only in jojoba, while putrescine did not affect rooting in either species or when using the different treatments (Table 2 ).
J o j o b a a n d apricot. -
TABLE I Effect of I mM putrescine ( + and - ), added to a double liquid layer, on leaf expansion observed at the end of the proliferation phase, and subsequent rooting percentage of the same explants in a rooting medium containing 5/~M NAA, lacking putrescine. (The signs --, + or --, - indicate the provenance of explants from double liquid layer with and without putrescine respectively) Explants with expanded leaves (%)
Control
+
-
-~+
--,-
-,+
--,-
Pear
77A*
35B
58.3A
80.0B
60.0A
53.3A
Chinese Pear, D6
10a
15a
26.6A
20.0A
66.6B
66.6B
Species
Rooting (%) Basal darkening
*Mean separation within line by Duncan's test, lowercase letters ( P = 0.05 ), uppercase letters (0.01 ).
68
E. RUGINI ET AL.
TABLE 2 Effect of basal shoot darkening on in vitro rooting of several woody species, with ( + ) or without ( - ) 1 mM putrescine, in a medium containing 5/iM NAA. Explants on auxin-free medium failed to root Species
Rooting (%) Control
Apple, M9 Walnut Chestnut Almond Jojoba Apricot Olive (apical explants) Olive (basal explants
23.2A* 0 0 0 20.0a 70.0a 66.6a 41.6a
Basal darkening +
-
+
50.0C 0 0 0 25.0a 66.6a 73.3a 78.3b
23.3A 38.3a 40.0a 60.0a 40.0b 68.3a 73.3a 75.0b
38.3B ! 0.0b 41.6a 58.3a 45.0b 65.0a 75.0a 80.0b
*Mean separation within line by Duncan's test, lowercase letters ( P - 0.05 ), uppercase letters (0.01 ).
In basal explants, both putrescine and basal darkening treatment increased rooting over the control (Table 2 ) and promoted an earlier root appearence (data not shown). Apical explants were not affected by the putrescine or by the basal shoot darkening treatments. Without any treatment (Control) they rooted in higher percentage (66.6%) than the basal ones (41.6% ) (signifiance not shown in Table 2 ). PCA-soluble free polyamines detected in three species (olive, chestnut and walnut) at the end of the shoot proliferation phase, were found to increase in shoots placed in the dark for a 10 day period as compared with shoots grown in a 16 h photoperiod. In all three species only traces of putrescine were detected, while spermidine was found in a low amount. High levels of spermidine/spermine were detected in walnut shoots. Total free PA were lowest in olive, intermediate in chestnut and highest in walnut shoots (Table 3). Throughout the first 8 days of rooting, PCA-soluble free, PCA-soluble bound and PCA-insoluble bound polyamines, detected in apical and basal olive explants, with both methods of rooting, showed an increase at Day 2 of PCAsoluble free and PCA-soluble bound (conjugated) content and then decreased in the subsequent days (data not shown). However, a greater difference was found among bound PA, only in the basal explants on Day 2 of rooting, where control explants showed a concentration of insoluble bound putrescine, spermidine/spermine, higher than those rooted with basal darkening (Table 4). Olive.
-
69
IN VITRO ROOTING IN WOODY SPECIES
TABLE 3 PCA-soluble free polyamine content in olive, chestnut and walnut shoots at the end of proliferation phase, in a 16-h photoperiod (L) and in the dark for 10 days (D) before excising the explants for rooting. Data are expressed in nmol (gFW)-~. Numbers represent means + SE oftwo samples Species
Putrescine
Spermidine
Spermine
Olive (L) Olive (D) Chestnut (L) Chestnut (D) Walnut (L) Walnut (D)
traces traces 10 + 2.0 10 + 0.5 traces traces
8 + 2.0 16 + 2.0 16 + 0.5 40 + 12.5 10 + 2.0 32 + 4.5
8 + 0.5 20 + 2.0 12 + 2.0 18 + 0.5 160 + 20 205 + 12.5
TABLE 4 PCA-insoluble bound polyamines in apical and basal explants of olive, detected at 0, 2, 5 and 8 days from the beginning of the rooting experiment, on Bourgin and Nitsch medium plus 5/~M NAA both with 16-h photoperiod (Control) and basal shoot darkening. Data are expressed ia nmol (gFW) The roots appeared after 12-15 days. Numbers represent means + SE of two samples Days
Apical explants Control
Putrescine
Spermidine
Spermine
Basal explants Basal darkening
Control
Basal darkening
0 2 5 8
0 0 0 0
0 0 0
0 58±!2.5 8±0 0
0 4±0 0
0 2 5 8
6±2 6±2 5±0.5 6±2
6±2 5±2 4±0.5
6±0.5 16±2 6±0.5 4±0.5
4±0.5 6±2 4±0.5
0 2 5 8
6±2 8±0.5 10±2 6±2
6±2 60±12.5 15±2 12±2
12±0.5 15±4.5 12±2
8±2 10±2 6±2
DISCUSSION AND CONCLUSION
The response of the shoots to exogenously applied PA in the rooting medium is still contradictory. In mung bean (Friedman et al., 1982) and in almond (Rugini and Wang, 1986 ) no response was obtained. Only an increase of roots per cutting in mung bean (Jarvis et al., 1983) and in English ivy (Geneve and Kester, 1991 ) has been observed; however, in chert3, at 0.5 mM, spermine decreased rooting (Biondi et al., 1990). Putrescine promoted ear-
70
E. RUGINI ET AL.
lier rooting in olive, both when rooting was induced by auxin or by Agrobacterium rhizogenes, and also increased rooting percentage of both in vitro explants and cuttings (Rugini et al. 1992). The reason for these different responses among the species, and sometimes among clones, must still be established. However, it could be that rooting depends on the endogenous level of PA. Our work shows that the different responses mainly depended on the species, environmental con&tions (such as basal shoot darkening), the type of explants and the endogenous PA content at the end of the proliferation phase, before using them for rooting. The content, in fact, seems to be inversely correlated with the response to exogenous PA treatment. In olive, in which putrescine treatment had a beneficial effect on rooting, the shoots contained very low endogenous free PA, both in those kept in the dark for the last l0 days of the proliferation phase and in those under a 16-h photoperiod (control). In walnut shoots that contained relatively high amounts of endogenous free PA, rooting was decreased by putrescine treatment, owing to the fact that the exogenous treatment contributed to overcoming the optimal endogenous PA concentration, by behaving as inhibitors. Chestnut, however, which contained an intermediate PA concentration between olive and walnut, was unaffected by putrescine treatment. The effect of dark on PA accumulation seems to be related to the shoot phase during proliferation or rooting. In the proliferation phase, the dark appears to allow for an accumulation of free spermidine and spermine. Only bound PA, particularly putrescine and spermine on Day 2, clearly accumulated during the rooting phase, but only in explants rooted under 16 h photoperiod. This fact might suggest a different role or utilization of PA during shoot growth, root induction and at different light conditions. The reason for this intense PA variation is still difficult to explain because little is known about the physiological significance of bound polyamines. Bound polyamines can bind with several substances and in several parts of the tissues (SerafiniFracassini and Mosetti, 1986; D'Orazi and Bagni, 1987). It is probable that in the dark, or in basal darkening, PA are degraded quicker. However it cannot be concluded that their catabolic products might also interfere with rooting induction. Very little is known about catabolism products and their specific roles. According to Torrigiani et al. (1987), free and bound putrescine and spermidine seem to be strongly involved in the root initiation phase in tobacco, as the level of PA rises immediately before root appearance. From our data, the spermine seems to interfere inste',d with the induction phase by subtracting the free PA and binding them on Day 2 which is a crucial period in the induction phase. This accumulation was noted only in basal shoots rooted in a 16-h photoperiod. Under these conditions, the shoots required putrescine treatment to improve their rooting. In apical explants and in those fr ~m the basal darkening treatments (both apical and basal), which did not require putrescine treatment to improve rooting, no bound PA accumulation
IN VITRO ROOTING IN WOODY SPECIES
71
was noted. In conclusion, the PA requirement for increasing rooting among the species may depend on two factors: ( 1 ) the content of free PA immediately before the rooting phase, or (2) the tendency to accumulate PA in bound form during the first 2 days when free PA are probably needed for root induction. This second hypothesis is strengthened by the observation that in apical olive explants and in explants rooted with the basal darkening treatment, putrescine treatment was not required for increasing or for promoting earlier rooting and bound PA accumulation was not detected. Therefore, further experiments are needed to clarify this aspect. ACKNOWLEDGEMENTS
Research supported by National Research Council of Italy, Special Project RAISA, Sub-project N. 2 Paper N514. We are grateful to Dr D.W. Fulbright for help in editing the paper and for useful comments. Thanks to Claudio Taratufolo for technical assistance in the laboratory and greenhouse and Vivai Vitrocoop and Battistini for supply some of cultures.
REFERENCES Biondi, S., Diaz, T., Iglesias, I., Gamberini, G. and Bagni N., 1990. Polyamines and ethylene in relation to adventitious root formation in Prunus avium shoot cultures. Physiol. Plant., 78: 474-483. Bourgin, J.P. and Nitsch, J.P., 1967. Obtention de Nicotiana haploides partir d' famines cuirives in vitro. Annu. Physiol. Veg., 9: 377-382. D'Orazi, D. and Bagni, N., 1987. In vitro interactions between polyamines and pectic substances. Biochem. Biophys. Res. Commun., 148:1259-1263. Driver, J.A. and Kuniyuki, A.H., 1984. In vitro propagation of Paradox walnut rootstock. HortScience., 19: 507-509. Druart, Ph., Kevers, C., Boxus, P.H. and Gaspar, T.H., 1982. In vitro promotion of root formation by apple shoots through darkness effect on endogenous phenols peroxidases. Z. Pflanzenphysiol., 5:429-436 Friedman, R., Altman, A. and Bachrach, U., 1982. Polyamines and root formation in mung bean hypocotyl cuttings. Effects of exogenous compounds and changes in endogenous polyamine content. Plant Physiol., 70: 844-848. Geneve, R.L. and Kester, S.T., 1991. Polyamines and adventitious root formation in the juvenile and mature phase of English ivy. J. Expl. Bot., 42:71-75. Jarvis, B.C., Shannon, P.R.M., and Yasmin, S., 1983. Involvement of polyamines with adventitious root development in stem cuttings of mung bean. Plant Cell Physiol., 24: 677-683. Lloyd, G. and McCown, B.H., 1981. Commercially feasible micropropagation of mountain laurel (Kahnia latifolia) by use of shoot tip culture. Comb. Proc. Int. Plant Prop. Soc., 30: 421427. Maene, L. and Debergh, P., 1985. Liquid medium additions to established tissue cultures to improve elongation in rooting in vivo. Plant Cell Tiss. Org. Cult., 5:23-33 Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-497.
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Nemeth, G., 1986. Induction of rooting. In: Y.P.S. Bajaj (Editor), Biotechnology in Agriculture and Forestry. Trees I. Springer, Berlin, pp. 49-64. Palavan, N and Galston, A.W., 1982. Polyamine biosynthesis and titer during various developmental stage ofPhaseolus vulgaris. Physiol. Plant., 55: 438-444. Rugini, E. and Fedeli, E., 1990. Olive (Oiea europaea L. ) as an oilseed crop. In: Y.P.S. Bajaj (Editor), Biotechnology in Agriculture and Forestry, Vol. 10, Springer, Berlin, pp. 593-641. Rugini, E.and Verma, D.C., 1983. Micropropagation of difficult to propagate almond (P. amygdalus Batch. ) cultivar. Plant Sci. Lett., 28:273-281. Rugini, E. and Wang, X.S., 1986. Effect of polyamines, 5-Azacytidine and growth regulators on rooting in vitro of fruit trees, treated and untreated with Agrobacterium rhizogenes. In; 6th Int. Congr. Plant Tissue Cell Culture University of Minnesota, Minneapolis, p. 374. Rugini, E., Jacoboni, A. and Bazzoffia, A., 1988. A simple in vitro method to avoid the initial dark period and to increase rooting in fruit trees. Acta Hortic., 227: 438-440. Rugini, E., Luppino M., De Agazio, M. and Grego, S., 1992. Endogenous polyamine and root morphogenesis variations under different treatments in cuttings and in in vitro explants of olive. Acta Hortic., 300: 225-232. Sankhla, N. and Upadhyaya, A., 1990. Polyamines and adventitious root formation, in: T.D. Davis, B.E. Hassig and N. Sankhla (Editors), Adventitious Root Formation in Cuttings. Dioscorides Press, Wishire, OR, pp. 202-209. Schenk, R.V. and Hildebrandt, A.C., 1972. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot., 50:199-204. Serafini-Fracassini, D. and Mosetti, U., 1986. What is the function of conjugated polyamines in plants? In: C.M. Caldarera, C. Clo' and C. Guarnieri (Editors), Biochemical Studies of Natural Polyamines. Cooperativa Libraria Universitaria Editori Bologna, Bologna, pp. 197202. Sriskandarajah, S., Mullins, M.G. and Nair, Y., 1982. Induction of adventitious rooting in vitro difficult-to-propagate cultivars of apple. Plant Sci. Lett., 24: 1-9. Torrigiani, P., Altamura, M.M., Pasqua, G., Monacelli, B., Serafini-Fracassini, D. and Bagni, N., 1987. Free and conjugated polyamines during de novo floral and vegetative bud tbrmation in thin cell layers of tobacco. Physiol. Plant., 70: 453-460. Von Arnold, S. and Erikson, T., 1984. Effect of agar concentration on growth and anatomy of adventitious shoots of Picea abies L. Karst. Plant Cell Tissue Organ Culture, 3: 257-264.
63
Role of basal shoot darkening and exogenous putrescine treatments on in vitro rooting and on endogenous polyamine changes in difficult-toroot woody species Eddo Rugini, Antonella Jacoboni and Maria Luppino Istituto di Ortofloroarboricoltura, Universitit della Tt,~cia, Facolti~ di Agraria, Via S. Camillo de Lellis, Ol lO0 Viterbo, Italy (Accepted 6 July 1992)
ABSTRACT Rugini, E., Jacoboni, A. and Luppino, M., 1993. Role of basal shoot darkening and exogenous putrescine treatments on in vitro rooting and on endogenous polyamine changes in difficult-to-root woody species. Scientia Hortic., 53: 63-72. The effect of in vitro putrescine treatment on several difficult-to-root fruit tree species was studied in order to improve rooting efficiency. Putrescine was added to a double liquid layer at the shoot proliferation phase and during the rooting phase. Shoots were rooted in either a i 6-h photoperiod or a 16-h photoperiod with shoot bases in the dark (basal darkening). Putrescine, at I mM, in a double liquid layer, increased the number of pear shoots with expanded leaf lamina but did not increase the rooting efficiency of these shoots. Putrescine increased the rooting of apple (M9 rootstock, clone P3 ) and the basal explants of olive, rooted in 16-h photoperiod; decreased it in walnut but did not affect chestnut, almond, jojoba and apricot rooting. Basal shoot darkening was essential for root formation in chestnut, walnut and almond. The endogenous content of perchloric acid (PCA)-soluble free polyamines (PA) was determined a~ the end of the proliferation phase in olive, chestnut and walnut. The shoots proliferated under the (~-h photoperiod contained a lesser concentration of free spermidine and spermine compared with mo~c kept in the dark for 10 days before being excised for rooting. High levels of spermine were detected in walnut shoots. Total free polyamine content was relatively low in olive, at an intermediate le,Jei in chestnut and high in walnut. The content seems to be inversely correlated with the response to exogenous putrescine treatments. In addition, PCA-soluble free and FCA-soluble bound polyamines in olive shoots, both basal and apical, either in a 16-h photoperiod or in basal darkening, showed an increase at Day 2 of rooting and then decreased in the following days. In 16-h photoperiods basal explants of olive, which under these conditions require applied exogenous putrescine for a strong rooting response, accumulated all three PA as bound forms at Day 2. However, those which rooted with basal shoot darkening did not respond to exogenous putrescine or accumulate PA. Keywords: almond; apricot; chestnut; Chinese pear; jojoba; olive; pear; walnut.
Correspondence to: E. Rugini, Instituto di Ortofloroarboricoltura, Universit/l della Tuscia, Facolt/~ di Agraria, Via S. Camillo de Lellis, 01100 Viterbo, Italy.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4238/93/$06.00
64
E. RUGINI ET AL.
Abbreviations: agar = agar purified (Sigma); BA = benzyladenine; DFMA = difluoromethylarginine; D F M O = difluoromethylornithine; GA3 = gibbereUic acid; 1BA = indolebutyric acid; NAA = alpha-naphthaleneacetic acid; PA = polyamine; PCA = perchloric acid.
INTRODUCTION
Rooting results from a complex of biochemical and physiological processes affected by several factors. Rooting in vitro is affected by genetics, juvenility, growth regulators, phenolic compounds, shoot quality of previous subcultures, peroxidase activity, photoperiod, light intensity and quality, etc. (Nemeth, 1986). In particular, it is well known that darkness during the first week of the rooting phase is often essential in stimulating rooting in several woody species (Druart et al., 1982; Sriskandarajah et al., 1982; Rugini and Verma, 1983), although, with dark pretreatment, survival of plantlets usually decreases when they are transferred to the soil (Von Arnold and Erikson, 1984). Darkening only the basal part ofolive and almond explants during the rooting phase, by painting the outside of the jar black and covering the medium surface with black polycarbonate granules, can replace the initial period of darkness required for an increase in rooting efficiency (Rugini et al., 1988 ). Among the several factors that can affect root induction, the endogenous polyamines (PA) may play an important role (Sankhla and Upadhyarya, 1990). The inhibition of PA synthesis by specific inhibitors such as diflouromethylarginine (DFMA) and difluoromethylornithin¢ (DFMO) inhibits rooting. This effect can be partially reversed by exogenous PA treatment (Biondi et al., 1990). The effect of exogenous PA treatment on rooting stimulation is not well understood and appears to be dependent on the plant species and the physiological state of the shoots before excising the explants or the plant before collecting the cuttings (Rugini et al., 1992). Exogenous PA treatment of olive, when combined with auxins, appears to promote earlier and higher rooting percentage ofboth in vitro explants and cuttings (Rugini and Wang, 1986). Therefore this work pursued two objectives: ( 1 ) to test the effect of exogenous putrescine on other fruit tree species; (2) to analyze the polyamine variation in olive during the first days of rooting, and in olive, chestnut and walnut shoots at the end of their proliferation phase. These species were chosen for their different responses to putrescine treatment, as putrescine positively affects olive rooting, but has no effect on chestnut rooting and decreases rooting in walnut. MATERIALS AND METHODS
Some difficult-to-root cultivars of the following species were used in this study: almond, apple, apricot, chestnut, Chinese pear rootstock, jojoba, olive,
IN VITRO ROOTING IN WOODY SPECIES
65
pear and walnut. With pear and Chinese pear rootstock, putrescine was added filter-sterile in the double liquid layer, which was incorporated on the solidified medium at the end of the culture cycle. Putrescine was added to the rooting medium for the other species. The following species, environmental conditions and media were adopted in this study: Pear (Pyrus communis L.), cultivar 'Kaiser', maintained on MS (Murashige and Skoog, 1962) medium plus 2.25 ~tM BA, 0.5/iM IBA, 3% sucrose and 0.7% agar with a double liquid layer and putrescine; Chinese pear rootstock (Pyrus calleryana), maintained on woody plant medium (WPM) salt medium (Lloyd and McCown, 1981 ) plus 0.66/zM BA, 0.08/zM NAA, 0.2 /~M GA3, 3% sucrose and 0.7% agar at pH 6, with a double liquid layer and with or without putrescine; walnut (Juglans regia L.), unknown ecotype, maintained on DKW (Driver and Kuniyuki, 1984) mineral salts medium plus 4.5/~M BA, 0.05/~M IBA, 3% sucrose and 0.7% agar, apple (Malus pumila), M9 rootstock, clone P3, maintained on MS basal salt medium plus 3.5 /iM BA, 3% sucrose and 0.7% agar at pH 5.8; chestnut (Castanea sativa Miller), maintained on DKW medium plus l ~tM BA, 3% sucrose and 0.7% agar; jojoba (Simmondsia chinensis (Link.) Schneider), maintained on MS medium plus 3.5/iM BA, 0.08/~M NAA, 3% sucrose and 0.7% agar; apricot (Prunus armeniaca L.), cultivar 'Bebeco', maintained on SH (Schenk and Hildebrandt, 1972) macroelements, MS microelements plus 3.5/~M BA, 3% sucrose and 0.7% agar; almond (Prunus dulcis Batsch), cultivar 'Ferraduel', maintained on DKW mineral salts plus 3.5/tM BA, 0.08/~M NAA, 3% sucrose and 0.7% agar, with 3-week subcultures; olive (Olea europaea L. ), cult:,var 'Moraiolo', maintained on olive (OM) medium (Rugini and Fedeli, 1990) plus 5/~M of zeatin. Olive, chestnut and walnut shoots were placed in the dark l 0 days prior to excising the microcuttings in order to verify how the dark was able to affect endogenous PA levels before the rooting phase. All shoots were derived from buds collected from l 0-15-year-old mature bearing plants, except for jojoba and walnut, which were derived from l-yearold and 5-year-old seedlings, respectively. All cultures were started at least 2 years earlier and maintained in 500 ml jars with 100 ml of its own proliferation medium and with 20 explants per jar. They were then placed in a growth room at 23°C +_ l °C, under fluorescent white cool light at 40/~mol m -2 s -~ with a 16-h photoperiod. The double liquid layer medium for pear and Chinese pear rootstock contained the mineral salts of MS plus 3% sucrose, l/zM BA and l/zM IBA at pH 5.5 with or without l mM putrescine. At the end of the culture cycle, 30 ml of the liquid medium was added to the agar solidified one as described by Maene and Debergh ( 1985 ), and left for 28 days; the shoot explants were then excised from cultures for the rooting test. Rooting was carried out in 120 mm × 16 mm test tubes with 5 ml Bourgin and Nitsch (1967) medium with macro elements reduced to half, plus 2% sucrose, with or without 5/~M NAA and 0.7% agar. One apical explant, 2-3
66
E. RUGINI ET AL.
cm long, was placed in each tube with a total of 60 explants for each treatment. The tubes were then placed in a growth room with the same environmental conditions used for the proliferation phase. The rooting experiment of olive, given its strong apical dominance, differed from the others because the entire shoot was divided into two parts (apical and basal) of equal length (two to three nodes) and kept separate. Apical explants were kept separated from the basal ones because, in our experience, putrescine only rarely affects rooting in olive apical explants in terms of early root promotion and increase of rooting percentage. The treatments, for all species, were as follows: ( 1) control ( 16-h photoperiod); (2) control with 1 mM of putrescine; (3) control with basal darkening; (4) control with basal darkening and with 1 mM of putrescine. The filter-sterilized putrescine was added, after autoclaving, before medium solidification. The black sterile polycarbonate granules (small cylinder of 2 mm in diameter and 2.5 mm high, from Montedison) were placed, before implanting the explants, on the surface of solidified medium in a layer of about 4 mm, in order to m~ke the medium dark. The outside of the tubes were painted black up to the medium level as previously described (Rugini et al., 1988). An additional sample of olive explants were prepared for endogenous polyamine determination. Rooting percentage was observed on the Day 28 and data were subjected to the analysis of variance, by subdividing the test tubes in four blocks of 15 tubes each. Polyamine extraction and analysis. - Two kinds of plant material were ana-
lyzed for PA content: ( 1) whole explants of olive at 0, 2, 5, and 8 days from the beginning of the rooting experiments; (2) olive, chestnut and walnut shoots, by discarding the basal part in contact with the medium, previously placed for 10 days in the dark, in comparison with the shoots grown in a 16h photoperiod. Each sample was subdivided into two repetitions of 0.5 g each and frozen in 2.5 ml of 5% cold perchloric acid (PCA). The samples were homogenized with Ultraturrax at a progressively increasing speed for about l min and centrifuged at 26000 × g for 10 min. The pellet was resuspended in 2.5 ml PCA and centrifuged again. A sample of 2.5 ml of the combined two supernatants and the pellet were separately hydrolyzed in 6 N HCI for 18 h at 110°C in vials. The hydrolysates were evaporated under vacuum and resuspended in the original volume of PCA. Aliquots of the PCA supernatant (free PA), of the same hydrolyzed supernatant (conjugated PA ) and of the hydrolyzed pellet (bound PA) were dansylated according to Palavan and Galston (1982) and Tonigiani et al. (1987) and separated by thin layer chromatography in chloroform:triethylamine (5:1), then detected with a spectrofluorimeter.
IN VITROROOTINGIN WOODYSPECIES
67
RESULTS
In all species the explants did not root without auxin addition both those treated or untreated with putrescine (data not shown ). Basal shoot darkening and putrescine affected rooting according to the species, type of explants and the endogenous PA content at the end of the proliferation phase. P e a r a n d C h i n e s e pear. - Most of the pear shoots, proliferated on a double liquid layer medium with putrescine, showed fully expanded leaves com~ pared with those without putrescine (Table 1 ). However, the microcuttings excised from these shoots, when placed on the rooting medium without putrescine, rooted in less percentage compared with those collected from a double liquid layer without putrescine. The basal darkening method seemed to affect, negatively, rooting of shoots proliferated without putrescine. However, in Chinese pear, putrescine added to a double liquid layer, did not affect leaf expansion or the rooting of the microcuttings, while basal darkening increased the rooting percentage of the explants (Table 1 ). Putrescine increased rooting percentage both in the control explants and in those with basal darkening. However, the basal darkening alone did not affect rooting (Table 2 ).
Apple. -
W a l n u t , c h e s t n u t a n d a l m o n d . - Rooting occurred only with the basal darkening method and putrescine had no effect on rooting except in walnut where rooting was significantly decreased by this treatment (Table 2 ).
The basal darkening treatment increased rooting over the control only in jojoba, while putrescine did not affect rooting in either species or when using the different treatments (Table 2 ).
J o j o b a a n d apricot. -
TABLE I Effect of I mM putrescine ( + and - ), added to a double liquid layer, on leaf expansion observed at the end of the proliferation phase, and subsequent rooting percentage of the same explants in a rooting medium containing 5/~M NAA, lacking putrescine. (The signs --, + or --, - indicate the provenance of explants from double liquid layer with and without putrescine respectively) Explants with expanded leaves (%)
Control
+
-
-~+
--,-
-,+
--,-
Pear
77A*
35B
58.3A
80.0B
60.0A
53.3A
Chinese Pear, D6
10a
15a
26.6A
20.0A
66.6B
66.6B
Species
Rooting (%) Basal darkening
*Mean separation within line by Duncan's test, lowercase letters ( P = 0.05 ), uppercase letters (0.01 ).
68
E. RUGINI ET AL.
TABLE 2 Effect of basal shoot darkening on in vitro rooting of several woody species, with ( + ) or without ( - ) 1 mM putrescine, in a medium containing 5/iM NAA. Explants on auxin-free medium failed to root Species
Rooting (%) Control
Apple, M9 Walnut Chestnut Almond Jojoba Apricot Olive (apical explants) Olive (basal explants
23.2A* 0 0 0 20.0a 70.0a 66.6a 41.6a
Basal darkening +
-
+
50.0C 0 0 0 25.0a 66.6a 73.3a 78.3b
23.3A 38.3a 40.0a 60.0a 40.0b 68.3a 73.3a 75.0b
38.3B ! 0.0b 41.6a 58.3a 45.0b 65.0a 75.0a 80.0b
*Mean separation within line by Duncan's test, lowercase letters ( P - 0.05 ), uppercase letters (0.01 ).
In basal explants, both putrescine and basal darkening treatment increased rooting over the control (Table 2 ) and promoted an earlier root appearence (data not shown). Apical explants were not affected by the putrescine or by the basal shoot darkening treatments. Without any treatment (Control) they rooted in higher percentage (66.6%) than the basal ones (41.6% ) (signifiance not shown in Table 2 ). PCA-soluble free polyamines detected in three species (olive, chestnut and walnut) at the end of the shoot proliferation phase, were found to increase in shoots placed in the dark for a 10 day period as compared with shoots grown in a 16 h photoperiod. In all three species only traces of putrescine were detected, while spermidine was found in a low amount. High levels of spermidine/spermine were detected in walnut shoots. Total free PA were lowest in olive, intermediate in chestnut and highest in walnut shoots (Table 3). Throughout the first 8 days of rooting, PCA-soluble free, PCA-soluble bound and PCA-insoluble bound polyamines, detected in apical and basal olive explants, with both methods of rooting, showed an increase at Day 2 of PCAsoluble free and PCA-soluble bound (conjugated) content and then decreased in the subsequent days (data not shown). However, a greater difference was found among bound PA, only in the basal explants on Day 2 of rooting, where control explants showed a concentration of insoluble bound putrescine, spermidine/spermine, higher than those rooted with basal darkening (Table 4). Olive.
-
69
IN VITRO ROOTING IN WOODY SPECIES
TABLE 3 PCA-soluble free polyamine content in olive, chestnut and walnut shoots at the end of proliferation phase, in a 16-h photoperiod (L) and in the dark for 10 days (D) before excising the explants for rooting. Data are expressed in nmol (gFW)-~. Numbers represent means + SE oftwo samples Species
Putrescine
Spermidine
Spermine
Olive (L) Olive (D) Chestnut (L) Chestnut (D) Walnut (L) Walnut (D)
traces traces 10 + 2.0 10 + 0.5 traces traces
8 + 2.0 16 + 2.0 16 + 0.5 40 + 12.5 10 + 2.0 32 + 4.5
8 + 0.5 20 + 2.0 12 + 2.0 18 + 0.5 160 + 20 205 + 12.5
TABLE 4 PCA-insoluble bound polyamines in apical and basal explants of olive, detected at 0, 2, 5 and 8 days from the beginning of the rooting experiment, on Bourgin and Nitsch medium plus 5/~M NAA both with 16-h photoperiod (Control) and basal shoot darkening. Data are expressed ia nmol (gFW) The roots appeared after 12-15 days. Numbers represent means + SE of two samples Days
Apical explants Control
Putrescine
Spermidine
Spermine
Basal explants Basal darkening
Control
Basal darkening
0 2 5 8
0 0 0 0
0 0 0
0 58±!2.5 8±0 0
0 4±0 0
0 2 5 8
6±2 6±2 5±0.5 6±2
6±2 5±2 4±0.5
6±0.5 16±2 6±0.5 4±0.5
4±0.5 6±2 4±0.5
0 2 5 8
6±2 8±0.5 10±2 6±2
6±2 60±12.5 15±2 12±2
12±0.5 15±4.5 12±2
8±2 10±2 6±2
DISCUSSION AND CONCLUSION
The response of the shoots to exogenously applied PA in the rooting medium is still contradictory. In mung bean (Friedman et al., 1982) and in almond (Rugini and Wang, 1986 ) no response was obtained. Only an increase of roots per cutting in mung bean (Jarvis et al., 1983) and in English ivy (Geneve and Kester, 1991 ) has been observed; however, in chert3, at 0.5 mM, spermine decreased rooting (Biondi et al., 1990). Putrescine promoted ear-
70
E. RUGINI ET AL.
lier rooting in olive, both when rooting was induced by auxin or by Agrobacterium rhizogenes, and also increased rooting percentage of both in vitro explants and cuttings (Rugini et al. 1992). The reason for these different responses among the species, and sometimes among clones, must still be established. However, it could be that rooting depends on the endogenous level of PA. Our work shows that the different responses mainly depended on the species, environmental con&tions (such as basal shoot darkening), the type of explants and the endogenous PA content at the end of the proliferation phase, before using them for rooting. The content, in fact, seems to be inversely correlated with the response to exogenous PA treatment. In olive, in which putrescine treatment had a beneficial effect on rooting, the shoots contained very low endogenous free PA, both in those kept in the dark for the last l0 days of the proliferation phase and in those under a 16-h photoperiod (control). In walnut shoots that contained relatively high amounts of endogenous free PA, rooting was decreased by putrescine treatment, owing to the fact that the exogenous treatment contributed to overcoming the optimal endogenous PA concentration, by behaving as inhibitors. Chestnut, however, which contained an intermediate PA concentration between olive and walnut, was unaffected by putrescine treatment. The effect of dark on PA accumulation seems to be related to the shoot phase during proliferation or rooting. In the proliferation phase, the dark appears to allow for an accumulation of free spermidine and spermine. Only bound PA, particularly putrescine and spermine on Day 2, clearly accumulated during the rooting phase, but only in explants rooted under 16 h photoperiod. This fact might suggest a different role or utilization of PA during shoot growth, root induction and at different light conditions. The reason for this intense PA variation is still difficult to explain because little is known about the physiological significance of bound polyamines. Bound polyamines can bind with several substances and in several parts of the tissues (SerafiniFracassini and Mosetti, 1986; D'Orazi and Bagni, 1987). It is probable that in the dark, or in basal darkening, PA are degraded quicker. However it cannot be concluded that their catabolic products might also interfere with rooting induction. Very little is known about catabolism products and their specific roles. According to Torrigiani et al. (1987), free and bound putrescine and spermidine seem to be strongly involved in the root initiation phase in tobacco, as the level of PA rises immediately before root appearance. From our data, the spermine seems to interfere inste',d with the induction phase by subtracting the free PA and binding them on Day 2 which is a crucial period in the induction phase. This accumulation was noted only in basal shoots rooted in a 16-h photoperiod. Under these conditions, the shoots required putrescine treatment to improve their rooting. In apical explants and in those fr ~m the basal darkening treatments (both apical and basal), which did not require putrescine treatment to improve rooting, no bound PA accumulation
IN VITRO ROOTING IN WOODY SPECIES
71
was noted. In conclusion, the PA requirement for increasing rooting among the species may depend on two factors: ( 1 ) the content of free PA immediately before the rooting phase, or (2) the tendency to accumulate PA in bound form during the first 2 days when free PA are probably needed for root induction. This second hypothesis is strengthened by the observation that in apical olive explants and in explants rooted with the basal darkening treatment, putrescine treatment was not required for increasing or for promoting earlier rooting and bound PA accumulation was not detected. Therefore, further experiments are needed to clarify this aspect. ACKNOWLEDGEMENTS
Research supported by National Research Council of Italy, Special Project RAISA, Sub-project N. 2 Paper N514. We are grateful to Dr D.W. Fulbright for help in editing the paper and for useful comments. Thanks to Claudio Taratufolo for technical assistance in the laboratory and greenhouse and Vivai Vitrocoop and Battistini for supply some of cultures.
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