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Adventitious rooting in chestnut: an anatomical investigation
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E LS EV I ER
HORTICULTuR~SCIENTIA Scientia Horticulturae 59 (1994) 197-205
Adventitious rooting in chestnut: an anatomical investigation S. Biricolti, A. Fabbri*, F. Ferrini, P.L. Pisani Dipartirnento di Ortoflorofrutticoltura, University of Florence, Via Donizetti, 6-50144 Florence, Italy
Accepted 6 May 1994
Abstract
The effects of girdling and etiolation on the anatomical structure and adventitious rooting of hybrid chestnut (Castanea sativa X Castanea crenata) cultivar 'Marigoule' shoots are reported and discussed. The treatments were: ( I ) shoot girdling (2-3 cm above stoolinsertion point ); (2) etiolation (covering stoolbed with soil ); (3) girdling and etiolation; (4) untreated control. Only the shoots of treatment (3) formed adventitious roots (70% rooting). Etiolation induced no substantial differences in shoot anatomy in comparison with the control, except for a greater accumulation of starch grains. The two girdling treatments stimulated cell division and growth, particularly evident in the swelling of the cortex just above the girdle, and the production of multi-seriate xylem rays, especially notable in treatment (3). This latter treatment also evinced diminished shoot-tissue differentiation compared with treatments (2) and (4), and poorly differentiated cortex sclerenchyma cells, which were arranged not as rings but in irregularly shaped groupings. Root primordia in the early formation stages were found next to the multi-seriate xylem rays in the youngest phloem of the treatment (3) shoots. These findings indicate that root formation occurs over a fairly lengthy period and is associated with anatomical changes in the involved shoot zones. The effects of etiolation and girdling in treatment (3) could not be separated, suggesting their synergistic influence on rooting in chestnut. Keywords: Castanea crenata X Castanea sativa; Etiolation; Girdling; Stoolbed propagation
* Corresponding author at: Istituto di Coltivazioni Arboree, University of Catania, Via Valdisavoia 5-95123 Catania, Italy. 0304-4238/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0304-4238 (94)00680-E
198
S. Biricolti et al. /Scientia Horticulturae 59 (1994) 197-205
1. Introduction
Chestnut is usually propagated by budding or grafting on seedlings. Completely asexual techniques would be a welcome improvement but propagation by cuttings has resulted in poor nursery performance. Stooling, which is used for commercial fruit species rootstocks, and to lesser extent cultivars, appears to be a promising technique. Its success in chestnut seems to depend on the application of growth regulators and, overall, on basal shoot girdling (Vieitez, 1955, 1956; Solignat, 1964; Vieitez, 1974; Vieitez and Vieitez, 1974; Caldwell and Mudge, 1985; Caldwell, 1986; Lagerstedt, 1987; Ferrini et al., 1992). The poor rooting ability of chestnut seems to stem from shoot anatomy as well as biochemical factors, a correlation between pronounced cortex sclerification and poor rooting of cuttings having been postulated (Vieitez and Vieitez, 1974). The sclerenchyma sheaths, which are found early in shoot growth, might act as mechanical barriers. It has been shown, however, that in this species too, the newly formed roots cause the disintegration of these tissues as their growth progress and the emergence of the roots themselves, which may appear to a certain extent hindered by the sclerenchyma rings, does in fact not seem to be adversely affected (Vieitez et al., 1980). The present study reports and discusses the changes induced in chestnut shoot structure by girdling and etiolation, and the resulting formation of adventitious roots in suckers, which received both these treatments.
2. Materials and methods
On 20 June 1990, 100 selected healthy stoolbeds of (Castanea sativa× Castanea crenata) cultivar 'Marigoule' were randomly subjected to: (1) shoot girdling; (2) etiolation; (3) girdling-etiolation; (4) untreated control. Girdling was performed with a plastic-covered wire 2-4 cm above the shoot-stool insertion point; etiolation of the lower part of the shoots was performed on the same day by covering the stoolbeds with 10-20 cm of soil. Rooting was assessed immediately after leaf fall (30 November) in all treatments. Anatomical investigations were performed on samples taken from shoots of average vigour on 23 May, 28 June, 25 July, 9 and 21 August, 3 and 25 September and 5 October. The samples consisted of the 4-5 cm portion above the girdle or in an analogous position in the control and etiolated treatments; the material was fixed in FAA and dehydrated in an ethyl alcohol series, then embedded in a prepolymerized resin, made of a mixture of butyl- and methyl-methacrylate (7:3). The 5 pm sections obtained with a Reichert-Jung 2040 semithin rotary microtome, were stained with either PAS (Periodic Acid-Shift's reagent) or Acid Fuchsin, and counterstained with Toluidine Blue (O'Brien and McCully, 1981 ).
S. Biricolti et al. /Scientia Horticulturae 59 (1994) 197-205
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3. Results
3.1. Rooting Only the shoots of the girdling-etiolation treatment produced roots (70% rooting) (Fig. 1 ), which were first found emerging immediately above the girdle on 3 September, 75 days after treatment. The two girdling treatments (1 and 3) induced hyperplasia of the shoots, which was usually more pronounced just above the girdle in the girdling alone treatment.
3.2. Anatomical features 3.2.1. Control Two sclerenchyma rings were visible by May in the cortex of the untreated shoots. The outer older ring was well differentiated and continuous, the inner younger one discontinuous. Continuous sclerification in both the xylem and phloem was noted throughout the growing season. A secondary, unevenly distributed thickening (reaction wood) can occur in the cell walls of the xylem (Esau, 1977 ). In the phloem, up to four sclerenchyma sheaths formed in addition to that of the pericycle (Fig. 2 ), the only one rich in sclereids and showing an uninterrupted ring. Xylem rays were all uniseriate. Phellogen activity became apparent only in October. Druses and prismatic crystals of calcium oxalate were found in the most recently formed phloem near differentiating fiber bundles.
Fig. 1. Rooting in a 'Marigoule' stoolbed. The suckers were girdled with a plastic-covered iron wire, and etiolated by covering with soil in mid-June.
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Fig. 2. Control, 25 September.Transversesectionof the cortex;four sclerenchymarings are visible (arrows). The mostdistal ring is composedof phloemfibersand sclereidsand is presentfromearly spring. Bar, 200/zm. 3.2.2. Etiolation t r e a t m e n t
Very few anatomical differences were noted in the etiolated shoots compared with the control. The most important seemed to be the more pronounced deposition activity of insoluble substances such as druses in the phloem, and polyphenols and starch in the medullary rays in etiolated shoots, especially in late summer-early autumn. 3.2.3. Girdling
The effects of girdling on shoot anatomy became visible in late July-early August ( 35-40 days after treatment), the rays in the recently produced xylem being multi-seriate and the pericycle beginning to swell. This swelling was caused by the onset of two distinct processes at two different sites in the cortex: one in the cells bordering internally and externally the pericycle ring, which produced large thin-walled cells with large druses and ample inter-cellular spaces, and the other in the outer pericycle cells (where the phellogen later formed), which produced smaller cells, often containing tannins and starch deposits, with smaller intercellular spaces but no druses. Only two sclerenchyma rings were found in most of the samples and, except for the poorly differentiated, irregularly arranged short fiber bundles, no sclerenchyma was produced for the rest of the season. In most samples, the outer pericycle ring was shattered by cellular outgrowth. Xylem activity following girdling was marked by the production of many parenchyma cells and of few vessels, which were often arranged tangentially in respect to the shoot axis; the cells of the multi-senate xylem rays contained polyphenolic inclusions, a feature that had not been found in the first two treatments. By October ( 103 days after treatment) the pericycle was completely crushed by the proliferating outer cortex, the polyphenols were abundant in both the phloem and pericycle
S. Biricolti et al. /Scientia Horticulturae 59 (1994.) 197-205
201
and most tissues were necrotic (Fig. 3 ). While the anomalies found in the xylem were at this time even more marked, the effects of girdling were only visible in swelling tissues, as the shoots in the 2-3 cm above the girdle had gradually resumed their normal anatomy in all the examined tissues. 3.2.4. Girdling-etiolation treatment Some of the anatomical changes found in the 'girdling only" treatment were also present in this one. The following comments are therefore confined to the most prominent changes, especially those associated with adventitious root formation. Eight days after treatment (28 June) shoot structure was only slightly affected, although the first signs of a reaction were visible, i.e. paired initials of rays appeared in the cambium and dividing cells were noticeable next to the pericycle sclerenchyma ring. One month later (27 July) xylem cell formation was more affected and numerous parenchyma cells had been formed by the cambium; the rays were often bi- and triseriate and made up of very large cells. The mitotic activity in the pericycle had continued, now extending to the sub-epidermal cells. While the area between the cambium and sclerenchyma was the site of intense cell division, no other rings were formed and in their place the cambium produced groups of poorly lignified fibers, irregularly distributed and at times very large (Fig. 4), as well as numerous crystal-rich parenchyma cells. The cambial zone became difficult to detect in many samples by late August: like that in the phloem-xylem transition area, cambium activity had become chaotic, often producing different types of cells in no discernible order. Adventitious root formation at this time was already advanced: both initials in the early stages of meristematic division (Fig. 5 ) and primordia at various growth stages were found (Figs.
Fig. 3. Girdling, 25 September. Typical parenchyma produced by the cambium (arrow) above the girdle. Bar, 100 pm.
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S. Biricolti et al. /Scientia Horticulturae 59 (1994) 197-205
Fig. 4. Girdling and etiolation, 21 August. Groups of poorly differentiated fibers appear at random within the phloem parenchyma; the most peripheral cells contain prismatic calcium oxalate crystals (arrows). Bar, 100/zm.
Fig. 5. Girdling and etiolation, 5 August. In at least four points (arrows), differentiating phloem cells have resumed mitotic activity, to form meristematic foci. Druses (arrows on white circle) are also present in the xylem parenchyma. (C) cambium. Bar, 50/~m. 6 and 7 ). Observation o f the smallest groups of initials put their origin among the recently formed and only slightly differentiated phloem cells; as these meristematic foci usually coincided with phloem rays, the first initials should be considered as undifferentiated phloem ray initials. The root primordia at the beginning consisted of only a few cells, which could be distinguished from surrounding tissues by their smaller size, thinner walls and their being bordered by druse cells; thereafter they lengthened and crossed the cortex tissues, their cells continuing to
S. Biricolti et aL / Scientia Horticulturae 59 (1994) 197-205
203
Fig. 6. Girdling and etiolation, 21 August. Early cell arrangement in a young primordium positioned next to a large xylem ray. Note border cells rich in druses (arrows). Bar, 100/lm.
Fig. 7. Girdling and etiolation, 21 August. An older and larger root primordium; differentiating vessels, with the new orientation (arrows), are already visible in the xylem (X). Bar, 100/~m.
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S. Biricolti et al. /Scientia Horticulturae 59 (1994) 197-205
i Fig. 8. Girdling and etiolation, 5 October. Root primordium emergence. The sclerenchyma (arrows) is completely shattered; most of the remaining cortex is necrotic and detached from the shoot. Bar, 500 #m.
divide and differentiate until arranging themselves in organized tissues before root emergence (Fig. 8).
4. Conclusion
Unlike etiolation, girdling induced marked histological alterations, at least in the area immediately above the girdle. The onset of the most pronounced changes, which coincided with the first reactions to girdling a few days after treatment, included interruption of the rhythmic production of sderenchyma rings, their replacement by less differentiated structures, the transition of the medullary rays from uniseriate to multi-senate, and the exceptional increase of parenchyma tissues in the hyperplastic cortex. Adventitious root formation occurred in the same area as that reported for etiolated plum shoots (Mittempergher, 1963) and for etiolated and hormone-sprayed chestnut shoots (Rinallo et al., 1987 ). It may be concluded from these results that anatomical conditions marked by numerous parenchyma and storage cells and a scarcity of sclerenchyma may promote the formation of new roots in chestnut shoots, which is consistent with the literature. However, these features are in our opinion also found in shoots that
S. Biricolti et al. / Scientia Horticulturae 59 (1994) 197-205
205
did not form root primordia (girdling) and, hence, cannot by themselves explain the event. Satisfactory rooting requires that girdling be followed by etiolation, which showed light anatomical effect when applied alone. This means that in the 'girdling-etiolation' treatment, biochemical effects, which cannot be studied microscopically, must combine with anatomical effects. This suggests, however, that an interesting approach to the problem would be the study of starch, and of some secondary products such as polyphenols and calcium oxalate in the root forming tissues. Acknowledgments The authors wish to thank AS.PRO.FRUT., Cuneo, for hosting this research in the Spinetta experimental field. The research was supported by a 40% MURST grant.
References Caldwell, B., 1986. Update on chestnut layering. 77th Annual Report of the Northern Nut Growers Association, pp. 116-122. Caldwell, B. and Mudge, K., 1985. Production of own rooted chestnut trees. 76th Annual Report of the Northern Nut Growers Association, pp. 92-97. Esau, K., 1977. Anatomy of Seed Plants. 2nd edn. John Wiley, pp. 138-139. Ferrini, F., Mattii, G.B., Nicese F.P. and Pisani, P.L., 1992. Effetti di trattamenti auxinici sulla radicazione in margotte di ceppaia di castagno. Atti Giornate Scientifiche SOl, Ravello 8-10 Aprile 1992, pp. 336-337. Lagerstedt, H.B., 1987. A review on chestnut propagation. In: M.S. Burnett and R.D. Wallace (Editors), Chestnuts and Creating a Commercial Industry. Proc. II Pacific Northwest Chestnut Congress, 22-23 August 1987, pp. 56-61. Mittempergher, L., 1963. Indagini sull'influenza dell'eziolamento sulla emissione di radici avventizie in alcuni susini. Riv. Ortoflorofrutt. Ital., 88 (2) :95-107. O'Brien, T.P. and McCully, M.E., 1981. The Study of Plant Structure. Principles and Selected Methods. Thermacarphi, Melbourne, Australia. Rinallo, C., Gellini, R. and Fabbri, A., 1987. Studies on rhizogenesis in Castanea sativa Mill. cuttings. Adv. Hortic. Sci., 1:27-33. Solignat, G., 1964. Rooting Chestnut Trees. 55th Annual Report of the Northern Nut Growers Association, pp. 33-36. Vieitez, A.M. and Vieitez, E., 1974. Effect of etiolation on rootability of Castanea sativa Mill. cuttings. Anal. Edaf. Agrobiol., 33:955-965. Vieitez, A.M., Ballester, A., Garcia, M.T. and Vieitez, E., 1980. Starch depletion and anatomical changes during the rooting of Castanea sativa Mill. cuttings. Sci. Hortic., 13:261-266. Vieitez, E., 1955. E1 empleo de sustancias de accion hormonal en el enraizamiento del castafio por acodo bajo. Anal. Edaf. Agrobiol., 14:483-518. Vieitez, E., 1956. Problemas que plantea el estaquillado del castafio. Anal. Edaf. Agrobiol., 15:629659. Vieitez, E., 1974. Vegetative propagation of chestnut. N.Z.J. For. Sci., 4 (2):242-252.
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E LS EV I ER
HORTICULTuR~SCIENTIA Scientia Horticulturae 59 (1994) 197-205
Adventitious rooting in chestnut: an anatomical investigation S. Biricolti, A. Fabbri*, F. Ferrini, P.L. Pisani Dipartirnento di Ortoflorofrutticoltura, University of Florence, Via Donizetti, 6-50144 Florence, Italy
Accepted 6 May 1994
Abstract
The effects of girdling and etiolation on the anatomical structure and adventitious rooting of hybrid chestnut (Castanea sativa X Castanea crenata) cultivar 'Marigoule' shoots are reported and discussed. The treatments were: ( I ) shoot girdling (2-3 cm above stoolinsertion point ); (2) etiolation (covering stoolbed with soil ); (3) girdling and etiolation; (4) untreated control. Only the shoots of treatment (3) formed adventitious roots (70% rooting). Etiolation induced no substantial differences in shoot anatomy in comparison with the control, except for a greater accumulation of starch grains. The two girdling treatments stimulated cell division and growth, particularly evident in the swelling of the cortex just above the girdle, and the production of multi-seriate xylem rays, especially notable in treatment (3). This latter treatment also evinced diminished shoot-tissue differentiation compared with treatments (2) and (4), and poorly differentiated cortex sclerenchyma cells, which were arranged not as rings but in irregularly shaped groupings. Root primordia in the early formation stages were found next to the multi-seriate xylem rays in the youngest phloem of the treatment (3) shoots. These findings indicate that root formation occurs over a fairly lengthy period and is associated with anatomical changes in the involved shoot zones. The effects of etiolation and girdling in treatment (3) could not be separated, suggesting their synergistic influence on rooting in chestnut. Keywords: Castanea crenata X Castanea sativa; Etiolation; Girdling; Stoolbed propagation
* Corresponding author at: Istituto di Coltivazioni Arboree, University of Catania, Via Valdisavoia 5-95123 Catania, Italy. 0304-4238/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0304-4238 (94)00680-E
198
S. Biricolti et al. /Scientia Horticulturae 59 (1994) 197-205
1. Introduction
Chestnut is usually propagated by budding or grafting on seedlings. Completely asexual techniques would be a welcome improvement but propagation by cuttings has resulted in poor nursery performance. Stooling, which is used for commercial fruit species rootstocks, and to lesser extent cultivars, appears to be a promising technique. Its success in chestnut seems to depend on the application of growth regulators and, overall, on basal shoot girdling (Vieitez, 1955, 1956; Solignat, 1964; Vieitez, 1974; Vieitez and Vieitez, 1974; Caldwell and Mudge, 1985; Caldwell, 1986; Lagerstedt, 1987; Ferrini et al., 1992). The poor rooting ability of chestnut seems to stem from shoot anatomy as well as biochemical factors, a correlation between pronounced cortex sclerification and poor rooting of cuttings having been postulated (Vieitez and Vieitez, 1974). The sclerenchyma sheaths, which are found early in shoot growth, might act as mechanical barriers. It has been shown, however, that in this species too, the newly formed roots cause the disintegration of these tissues as their growth progress and the emergence of the roots themselves, which may appear to a certain extent hindered by the sclerenchyma rings, does in fact not seem to be adversely affected (Vieitez et al., 1980). The present study reports and discusses the changes induced in chestnut shoot structure by girdling and etiolation, and the resulting formation of adventitious roots in suckers, which received both these treatments.
2. Materials and methods
On 20 June 1990, 100 selected healthy stoolbeds of (Castanea sativa× Castanea crenata) cultivar 'Marigoule' were randomly subjected to: (1) shoot girdling; (2) etiolation; (3) girdling-etiolation; (4) untreated control. Girdling was performed with a plastic-covered wire 2-4 cm above the shoot-stool insertion point; etiolation of the lower part of the shoots was performed on the same day by covering the stoolbeds with 10-20 cm of soil. Rooting was assessed immediately after leaf fall (30 November) in all treatments. Anatomical investigations were performed on samples taken from shoots of average vigour on 23 May, 28 June, 25 July, 9 and 21 August, 3 and 25 September and 5 October. The samples consisted of the 4-5 cm portion above the girdle or in an analogous position in the control and etiolated treatments; the material was fixed in FAA and dehydrated in an ethyl alcohol series, then embedded in a prepolymerized resin, made of a mixture of butyl- and methyl-methacrylate (7:3). The 5 pm sections obtained with a Reichert-Jung 2040 semithin rotary microtome, were stained with either PAS (Periodic Acid-Shift's reagent) or Acid Fuchsin, and counterstained with Toluidine Blue (O'Brien and McCully, 1981 ).
S. Biricolti et al. /Scientia Horticulturae 59 (1994) 197-205
199
3. Results
3.1. Rooting Only the shoots of the girdling-etiolation treatment produced roots (70% rooting) (Fig. 1 ), which were first found emerging immediately above the girdle on 3 September, 75 days after treatment. The two girdling treatments (1 and 3) induced hyperplasia of the shoots, which was usually more pronounced just above the girdle in the girdling alone treatment.
3.2. Anatomical features 3.2.1. Control Two sclerenchyma rings were visible by May in the cortex of the untreated shoots. The outer older ring was well differentiated and continuous, the inner younger one discontinuous. Continuous sclerification in both the xylem and phloem was noted throughout the growing season. A secondary, unevenly distributed thickening (reaction wood) can occur in the cell walls of the xylem (Esau, 1977 ). In the phloem, up to four sclerenchyma sheaths formed in addition to that of the pericycle (Fig. 2 ), the only one rich in sclereids and showing an uninterrupted ring. Xylem rays were all uniseriate. Phellogen activity became apparent only in October. Druses and prismatic crystals of calcium oxalate were found in the most recently formed phloem near differentiating fiber bundles.
Fig. 1. Rooting in a 'Marigoule' stoolbed. The suckers were girdled with a plastic-covered iron wire, and etiolated by covering with soil in mid-June.
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S. Biricolti et al. / Scientia Horticulturae 59 (1994) 197-205
Fig. 2. Control, 25 September.Transversesectionof the cortex;four sclerenchymarings are visible (arrows). The mostdistal ring is composedof phloemfibersand sclereidsand is presentfromearly spring. Bar, 200/zm. 3.2.2. Etiolation t r e a t m e n t
Very few anatomical differences were noted in the etiolated shoots compared with the control. The most important seemed to be the more pronounced deposition activity of insoluble substances such as druses in the phloem, and polyphenols and starch in the medullary rays in etiolated shoots, especially in late summer-early autumn. 3.2.3. Girdling
The effects of girdling on shoot anatomy became visible in late July-early August ( 35-40 days after treatment), the rays in the recently produced xylem being multi-seriate and the pericycle beginning to swell. This swelling was caused by the onset of two distinct processes at two different sites in the cortex: one in the cells bordering internally and externally the pericycle ring, which produced large thin-walled cells with large druses and ample inter-cellular spaces, and the other in the outer pericycle cells (where the phellogen later formed), which produced smaller cells, often containing tannins and starch deposits, with smaller intercellular spaces but no druses. Only two sclerenchyma rings were found in most of the samples and, except for the poorly differentiated, irregularly arranged short fiber bundles, no sclerenchyma was produced for the rest of the season. In most samples, the outer pericycle ring was shattered by cellular outgrowth. Xylem activity following girdling was marked by the production of many parenchyma cells and of few vessels, which were often arranged tangentially in respect to the shoot axis; the cells of the multi-senate xylem rays contained polyphenolic inclusions, a feature that had not been found in the first two treatments. By October ( 103 days after treatment) the pericycle was completely crushed by the proliferating outer cortex, the polyphenols were abundant in both the phloem and pericycle
S. Biricolti et al. /Scientia Horticulturae 59 (1994.) 197-205
201
and most tissues were necrotic (Fig. 3 ). While the anomalies found in the xylem were at this time even more marked, the effects of girdling were only visible in swelling tissues, as the shoots in the 2-3 cm above the girdle had gradually resumed their normal anatomy in all the examined tissues. 3.2.4. Girdling-etiolation treatment Some of the anatomical changes found in the 'girdling only" treatment were also present in this one. The following comments are therefore confined to the most prominent changes, especially those associated with adventitious root formation. Eight days after treatment (28 June) shoot structure was only slightly affected, although the first signs of a reaction were visible, i.e. paired initials of rays appeared in the cambium and dividing cells were noticeable next to the pericycle sclerenchyma ring. One month later (27 July) xylem cell formation was more affected and numerous parenchyma cells had been formed by the cambium; the rays were often bi- and triseriate and made up of very large cells. The mitotic activity in the pericycle had continued, now extending to the sub-epidermal cells. While the area between the cambium and sclerenchyma was the site of intense cell division, no other rings were formed and in their place the cambium produced groups of poorly lignified fibers, irregularly distributed and at times very large (Fig. 4), as well as numerous crystal-rich parenchyma cells. The cambial zone became difficult to detect in many samples by late August: like that in the phloem-xylem transition area, cambium activity had become chaotic, often producing different types of cells in no discernible order. Adventitious root formation at this time was already advanced: both initials in the early stages of meristematic division (Fig. 5 ) and primordia at various growth stages were found (Figs.
Fig. 3. Girdling, 25 September. Typical parenchyma produced by the cambium (arrow) above the girdle. Bar, 100 pm.
202
S. Biricolti et al. /Scientia Horticulturae 59 (1994) 197-205
Fig. 4. Girdling and etiolation, 21 August. Groups of poorly differentiated fibers appear at random within the phloem parenchyma; the most peripheral cells contain prismatic calcium oxalate crystals (arrows). Bar, 100/zm.
Fig. 5. Girdling and etiolation, 5 August. In at least four points (arrows), differentiating phloem cells have resumed mitotic activity, to form meristematic foci. Druses (arrows on white circle) are also present in the xylem parenchyma. (C) cambium. Bar, 50/~m. 6 and 7 ). Observation o f the smallest groups of initials put their origin among the recently formed and only slightly differentiated phloem cells; as these meristematic foci usually coincided with phloem rays, the first initials should be considered as undifferentiated phloem ray initials. The root primordia at the beginning consisted of only a few cells, which could be distinguished from surrounding tissues by their smaller size, thinner walls and their being bordered by druse cells; thereafter they lengthened and crossed the cortex tissues, their cells continuing to
S. Biricolti et aL / Scientia Horticulturae 59 (1994) 197-205
203
Fig. 6. Girdling and etiolation, 21 August. Early cell arrangement in a young primordium positioned next to a large xylem ray. Note border cells rich in druses (arrows). Bar, 100/lm.
Fig. 7. Girdling and etiolation, 21 August. An older and larger root primordium; differentiating vessels, with the new orientation (arrows), are already visible in the xylem (X). Bar, 100/~m.
204
S. Biricolti et al. /Scientia Horticulturae 59 (1994) 197-205
i Fig. 8. Girdling and etiolation, 5 October. Root primordium emergence. The sclerenchyma (arrows) is completely shattered; most of the remaining cortex is necrotic and detached from the shoot. Bar, 500 #m.
divide and differentiate until arranging themselves in organized tissues before root emergence (Fig. 8).
4. Conclusion
Unlike etiolation, girdling induced marked histological alterations, at least in the area immediately above the girdle. The onset of the most pronounced changes, which coincided with the first reactions to girdling a few days after treatment, included interruption of the rhythmic production of sderenchyma rings, their replacement by less differentiated structures, the transition of the medullary rays from uniseriate to multi-senate, and the exceptional increase of parenchyma tissues in the hyperplastic cortex. Adventitious root formation occurred in the same area as that reported for etiolated plum shoots (Mittempergher, 1963) and for etiolated and hormone-sprayed chestnut shoots (Rinallo et al., 1987 ). It may be concluded from these results that anatomical conditions marked by numerous parenchyma and storage cells and a scarcity of sclerenchyma may promote the formation of new roots in chestnut shoots, which is consistent with the literature. However, these features are in our opinion also found in shoots that
S. Biricolti et al. / Scientia Horticulturae 59 (1994) 197-205
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did not form root primordia (girdling) and, hence, cannot by themselves explain the event. Satisfactory rooting requires that girdling be followed by etiolation, which showed light anatomical effect when applied alone. This means that in the 'girdling-etiolation' treatment, biochemical effects, which cannot be studied microscopically, must combine with anatomical effects. This suggests, however, that an interesting approach to the problem would be the study of starch, and of some secondary products such as polyphenols and calcium oxalate in the root forming tissues. Acknowledgments The authors wish to thank AS.PRO.FRUT., Cuneo, for hosting this research in the Spinetta experimental field. The research was supported by a 40% MURST grant.
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