- Email: [email protected]
Occurrence of Lenticels in Roots of Scots Pine Seedlings in Different Growth Conditions
f. Plant PhysioL Vol. 143. pp. 325-329 (1994)
Occurrence of Lenticels in Roots of Scots Pine Seedlings in Different Growth Conditions TUIJA
S. ARONEN and HELY M. HXGGMAN
The Finnish Forest Research Institute, Punkaharju Research Station, SF-58450 Punkaharju, Finland Received August 17, 1993 · Accepted October 20, 1993
Summary Lenticel tissues were observed in the roots of Scots pine seedlings at different oxygen poor growth conditions: hydroponic culture, in vitro culture, peatland and potted seedlings in the greenhouse. In the roots of seedlings from the typical heath forest site, lenticel formation could not be found. Anatomically, the lenticels consisted of filling cells above the expanded phelloderm region. In peatland seedlings, closing layers were also detectable. The lenticels might be beneficial to the Scots pine at wet growth habitats for gas exchange, even though their proliferation is too slow to be advantageous in flooding incidents.
Key words: Pinus sylvestris, Scots pine, lenticel, roots. Introduction Lenticels are structurally differentiated portions of the periderm characterized by a relatively loose arrangement of cells with intercellular spaces. They exist on almost all longstanding organs like roots and stems, and they have been found both in gymnosperms and in deciduous tree species (Devaux, 1900; Wutz, 1955; Esau, 1965; Buvat, 1989). Structural characteristics of lenticels, like continuity of intercellular spaces with interior parts of stem or root, were interpreted to indicate their functional role in gas exchange (Esau, 1965; Buvat, 1989). This was confirmed in the case of the deciduous tree, Nyssa sylvatica, (Hook et al., 1971), as well as in several coniferous species, e.g. Pinus contorta (Philipson and Coutts, 1978, 1980), Pinus clausa, P. serotina and P. taeda (Topa and McLeod, 1986 b). In hydroponic P. seratina and P. taeda seedlings, stem and root collar lenticels were the major sites of atmospheric 0 2 entry for roots (Topa and McLeod, 1986 b). Scots pine, the economically most important tree species in Finland, is light demanding and grows in different soil types throughout the whole country (Hannelius et al., 1989). The root system and root morphology (Laitakari, 1927) as well as several aspects of mycorrhizal development (e.g. Laiho, 1965; Stenstrom, 1990; Sarjala, 1991) in Scots pine, Pinus sylvestris, have been studied extensively. The existence of lenticels in Scots pine roots has, however, not been reported © 1994 by Gustav Fischer Verlag, Stuttgart
so far. In the present study the development and structure of lenticel tissues have been observed in roots of Scots pine seedlings. The effect of different growth conditions and seed~ing .age on lenticel formation has been studied in vitro and tn
vwo.
Material and Methods Scots pine (Pinus sylvestris L.) seedlings used in the present study were 1) collected from natural stands, 2} potted seedlings in the greenhouse, 3) hydroponic seedlings, and 4) seedlings grown in vitro. Scots pine seedlings were collected from natural stands in Punkaharju (61 o 48' N; 29° 17' E) and in Mekrijarvi (62° 47' N; 30° 57' E). In Punkaharju, 1- to 5-year-old seedlings (n = 29) were collected from the Vaccinium type (VT) site belonging to the dry and dryish heath forest class (Cajander, 1949), where Scots pine stands grow well and are of high quality. In Mekrijarvi, 3- to 10year-old seedlings (n • 26) were collected from a very wet site belonging to the pine peat-moor class (Cajander, 1949), where Scots pines are stunted and of low productivity. The seedlings were spaded with the soil surrounding the roots. In the laboratory the soil was carefully removed under tap water. One- and two-year-old potted seedlings were established with seeds from a local (61 ° 48' N; 29° 17' E) natural stand. The 1-yearold seedlings (n = 50) were grown in containers 8 em in diameter and the 2-year-old ones (n = 50) in containers 13 em in diameter.
326
TUIJA S. ARoNEN and Hm.Y M. HAGGMAN
The hydroponic seedlings (n - 44) were established with the same local seed as the potted ones. They were grown in pots for 4 months and then transplanted into pure solution culture. The seedlings were grown in plastic boxes in the greenhouse, seven seedlings in each 8-L box. The culture solution, replaced completely once a week, included 0.06% commercial fertilizer 5-Superex (Kekkila) containing 10.9% N, 4.0% P, 25.3% K and 1.5% Mg. The solution was aerated by using pressurized air 0.1 Llmin per 1L of culture solution. The 1-year-old in vitro seedlings (n = 378) were established with the local forest seed collected from a natural stand in Punkaharju (61 ° 48' N; 29° 17' E). The surface sterilized (3% Ca-hypochloride) seeds were sown and seedlings grown on ~ GD-media (Gresshoff and Doy, 1972), solidified with 1% agar and without any growth regulators in glass jars 4.5 em in diameter. The seedlings were kept at 23 °C in a 16: 8 h light-dark cycle. The seedlings, with the rest of the old media still surrounding the roots, were transferred into the new jars with fresh media every second month. Occurrence of lenticels in the roots of the seedlings was examined both visually and by using a dissecting microscope. The anatomical structure of lenticels was studied by using a research microscope. Roots with lenticel tissues were fixed in FAA (formalin:asetic acid:95% ethanol, 10:5:85, v/v/v), embedded in paraffm and stained with safranin-fast green according to Gerlach (1984). Surface structures of lenticels were studied by using a scanning electrone microscope (SEM) JEOL JSM-820. The glutaraldehyde (6.25 %)-fixed material was dehydrated in an alcohol gradient, dried by the Critical Point method, mounted on SEM adapters with double-sided adhesive tape and coated with gold.
...
Results and Discussion The different growth conditions remarkably affected the growth habit of the Scots pine seedlings and their roots {Fig. 1). At the typical heath forest site the seedlings had a strong taproot with several lateral roots {Fig. 1 D, 1 E, 1 F). The roots of the peatland seedlings were short and poorly branched. Both the hydroponic {Fig. 1 B) and the potted seedlings {Fig. 1 C) developed large and well branched root systems with taproots. In all of these seedling types the roots were covered with well developed cork. The in vitro seedlings {Fig. 1 A) had no distinguishable taproot, but did have a few slender roots. These roots were covered mostly by epidermis, which in older parts was replaced by thin cork. Lenticel tissues were observed in the roots of Scots pine in different growth conditions: hydroponic culture, in vitro culture, peatland and potted seedlings in the greenhouse. In the roots of seedlings from the typical heath forest site lenticel formation could not be found. In the roots of the potted seedlings the lenticels were small, hardly visible when compared with the size of the lenticels in the seedlings from hydroponic culture, in vitro culture and peatland {Fig. 2). The powdery, pale yellow lenticel tissue appeared as patches protruding from one to several millimeters from the root surface (Fig. 3). In several cases the lenticels were located either beside or around {Fig. 2 D) the emerging lateral roots. The appearance and the amount of the lenticels in Scots pine roots varied according to the growth conditions (Table 1). In the hydroponic seedlings the first lenticels could be observed after 1 month of culture, and after 2 months they
Fig.1: Different root types of Scots pine seedlings developed under different growth conditions. A. In vitro seedling. B. Seedling in hydroponic culture. C. Potted seedling in the greenhouse. D., E., and F. Two, three, and five-year-old heath forest site seedlings, respectively.
could be found in 42% of the seedlings. In in vitro seedlings the first observations of the developing lenticels were made after 2 months of culture. In the potted seedlings, on the other hand, remarkable amounts of lenticels did not appear until the age of 2 years. In the 3- to 10-year-old peatland seedlings, considerable amounts of the lenticels were also detectable. Unfortunately, very young seedlings could not be found at the particular peatland site. The highest amount of
Lenticels in pine roots
,......
..
·~·
t
r
~-------
• :L ··: .:·.::::::-~-:~----·-=--.-.. -..... - :· ·- ····-··----.....- - - -
327
.
,~
'if., ,.;J&t· ~
...
.,..
. . -.-.. -.. .c.-. . . -.. .,. .
-~----
;-.o .........-~--·---·-- •. ,
./1.·J
Fig. 2: Proliferation of lenticels in the roots of Scots pine seedlings. A. In vitro seedling growing on tissue culture media. B. Potted seedling in the greenhouse. Lenticel (arrow) formation in the taproot. C. Seedling in hydroponic culture with remarkable lenticel formation. D. Seedling growing at a very wet site in peatland. Lenticel around the emerging lateral root. Scale bars 3 mm.
Table 1: Occurrence of lenticels in roots of different types of Scots pine seedlings. + = low amount of lenticels; + + = moderate amount of lenticels; + + + = high amount of lenticels. Seedling type
Seedling age (years)
in vitro
n- 378
Fig. 3: Scanning electron micrograph presenting the surface structure of a lenticel in the roots of a Scots pine in vitro seedling.
Occurrence of lenticels in roots of seedlings studied
Amount of lenticels per seedling
54
++
(%)
hydroponic n = 44 n- 44
2 months 1 7 months 1
42 95
+++ +++
potted n =50 n • 50
1 2
4 44
+ ++
heath forest n • 11 n- 14 n=4
1-2 3 4-5
0 0 0
peatland n • 26
3-10
46
1
Months in hydroponic culture.
++
328
TUJJA S. ARoNEN and Hm.Y M. HXGGMAN
lenticels per seedling was found in hydroponic culture, where the roots were almost covered with lenticel tissues. In in vitro, peatland and 2-year-old potted seedlings the number of lenticels per seedling varied from a few single ones up to several dozens. In 1-year-old potted seedlings the lenticel number was lowest, only one or two per seedling. All of the growth conditions, inducing lenticel formation, could be considered as oxygen poor environments. In typical peatland sites, according to Hannelius and co-workers (1989), Scots pine seedlings suffer from deficiency of air space together with minor oxygen content in soil. Hydroponic culture systems have also earlier been used to imitate anaerobic environments (Topa and McLeod, 1986 a). The in vitro grown Scots pine seedlings have been observed to grow better with a continuous supply of air (Flygh et al., 1988). In potted seedlings the formation of smalllenticels after 2 years could be due to the shortage of oxygen caused by the excess of roots in a limited space (plastic pot). The lenticels were abundant in old cork covered roots of all seedling types, but they were also recognizable in young organs, like in epidermis covered roots, especially in hydro-
ponic and in vitro seedlings. This phenomenon was already described by Esau (1965) and Buvat (1989). Topa and McLeod (1986a) reported remarkable lenticel formation in pond and loblolly pines after 30 days of hydroponic culture, but in the case of sand pine the lenticel formation was nearly absent. According to our results considerable amounts of lenticels in Scots pine appeared much later than 1 month. Thus, the appearance of lenticels in Scots pine resembles more that of sand pine than that of pond or loblolly pines. Anatomically, lenticel tissues in Scots pine roots consisted of filling cells arising from repited cell divisions in phellogen. Filling cells were of angular form, more or less elongated (Fig. 4 B, 4 C). Intercellular air spaces between filling cells expanded towards the surface of the lenticels (Fig. 4 A, 4 B). Closing layers in lenticels were found only in the roots of the peatland seedlings (Fig. 4 B). In the lenticel regions phelloderm was usually expanded, consisting of several layers of compactly arranged cells (Fig. 4 A, 4 C). This was particularly true in the case of in vitro seedlings, in which the lenticels had only a limited number of the filling cells (Fig. 4 A). Throughout the pericycle region intercellular air
..
·"'"'
··~
...
..
. . ~ ,, .
,·'
· ..
..
Fig. 4: Anatomical characteristics of lenticels in the roots of Scots pine seedlings. A. Anatomical structure of a lenticel on the root of an in vitro seedling. B. A lenticel of a peatland seedling with characteristic closing cell layers and abundant amounts of filling cells. C. Expanded phelloderm region in the lenticel of an in vitro seedling. D. Intercellular air spaces throughout the pericycle region of an in vitro seedling. Scale bar in A. and B. 500 J.lm and in C. and D. 100 J.lm. Abbreviations: CL, closing layer; CW, cell wall fragments; FC, filling cells; IS, intercellular air spaces; Pc, pericycle; Pd, phelloderm; Pg, phellogen; X, xylem.
Lenticels in pine roots
spaces were abundant, extending from the phelloderm to the phloem {Fig. 4D). The cells of the pericycle typically contained a lot of starch grains {Fig. 4 D). The air spaces in the pericycle appeared to be predominantly lysigenous due to cell wall fragments present in aerenchyma. Lenticels in the roots of conifers have been studied by Topa and McLeod ( 1986 a, 1986 b). They could not find any closing cells in lenticels of pond and loblolly pines, or expanded phelloderm regions under the filling cells. They also reported that the intercellular air spaces in the pericycle of anaerobically grown pine seedlings were particularly prominent adjacent to a lenticel. These anatomical differences between the results of the present study and those of T opa and McLeod's could be due to different durations of the oxygenpoor growth conditions, different age of the seedlings, or different pine species. In other respects the anatomical structure of Scots pine lenticels corresponded with that described earlier in pond and loblolly pine (Topa and McLeod, 1986 b). Expanded phelloderm regions, such as we found in Scots pine, have also been reported with other species like Coriaria myrtifolia (Devaux, 1900). Lenticels with closing layers have been described in many decidious tree species already by Devaux (1900), but until the present study they have not been found in coniferous species. The abundance of intercellular air spaces in the Scots pine roots could indicate adaptation to anaerobic growth conditions as described earlier in angiosperms by Davy and co-workers (1990), Jackson (1990) and Peterson {1992). According to the present results the oxygen poor growth conditions cause anatomical adaptations in Scots pine roots. Previous investigations have revealed that the ability of different pine species to form lenticels correlates to their flooding tolerance, being abundant in flooding tolerant species (Topa and McLeod, 1986a). Our results show that the flooding intolerant .Scots pine (Hannelius et al., 1989) is able to form remarkable amounts of lenticels. The lenticel proliferation in Scots pine roots, however, seems to take too long to be beneficial in flooding incidents. Although Scots pine can not survive flooding, it is common in wet growth habitats like in peatlands (Paavilainen and Tiihonen, 1988), where the lenticels might be advantageous. Acknowledgements We are grateful to Dr. Anneli Kauppi for critical reading of the manuscript. We would also like to thank Ms. Rauni Ritola, Ms. Rauni Kantola and Mr. Jouko Lehto for technical assistance during the work.
References BuvAT, R.: Ontogeny, cell differentiation and structure of vascular plants, 2nd ed. Springer-Verlag, Berlin, Heidelberg (1989).
329
CAJANDEll, A. K.: Forest types and their significance. Acta For. Fenn. 56, 1-71 (1949). DAVY, A. J., S.M. NoBLE, and R. P. OuVEll: Genetic variation and adaptation to flooding in plants. Aquat. Bot. 38, 91-108 (1990). DEvAux, H.: Recherches sur les lenticelles. Ann. des Sci. Nat., Bot. Ser. 8. 12, 1-240 (1900). ESAu, K.: Plant Anatomy, 2nd ed. John Wiley & Sons, New York (1965). FLYGH, G., G. GllONRoos, and S. voN AllNoLD: Optimizing growth of conifer seedlings in vitro. Physiol. Plant. 73 (2), 20 A (1988). GEllLAcH, D.: Botanische Mikrotechnik. George Thieme Verlag, Stuttgart (1984). GllESSHOFF, P. M. and C. DoY: Development and differentiation of haploid Lycopersicon esculentum (Tomato). Planta 107, 161-170 (1972). HANNE:uus, S., M. LmKoLA, and A. TUIMALA: Metsakirja, metsanomistajan kasikirja. WSOY, Porvoo (1989). HooK, D. D., C. L. BlloWN, and P. P. KollMANIK: Inductive flood tolerance in swamp tupelo (Nyssa sylvatica var. biflora (Walt.) Sarg.).J. Exp. Bot. 22,78-89 (1971). jACKSON, M. B.: Hormones and developmental change in plants subjected to submergence or soil waterlogging. Aquat. Bot. 38, 4972 (1990). LAnio, 0.: Further studies on the ectendotrophic mycorrhiza. Acta For. Fenn. 79, 1-35 (1965). LArrAKAlll, E.: The root system of pine (Pinus sylvestris). A morphological investigation. Acta For. Fenn. 33, 307-380 (1927}. PAAVILAINEN, E. and P. TITHONEN: Peatland forests in Finland in 1951-1984. Folia Forestalia 714, 1-29 (1988). PETEllSON, R. L.: Adaptations of root structure in relation to biotic and abiotic factors. Can. J. Bot. 70, 661-675 (1992). PHIUPSON, J. J. and M.P. CouTTs: The tolerance of tree roots to waterlogging. III. Oxygen transport in lodgepole pine and Sitka spruce roots of primary structure. New Phytol. 80, 341-349 (1978). - - The tolerance of tree roots to waterlogging. IV. Oxygen transport in lodgepole pine and Sitka spruce roots of primary structure. New Phytol. 85, 489-494 (1980). SARJALA, T.: Nitrogen assimilation in ectomycorrhizal fungi and mycorrhizal and non-mycorrhizal Scots pine seedlings. Acta Univ. Oul. A218 (1991}. STENsnOM, E.: Ecology of mycorrhizal Pinus sylvestris seedlings aspects of colonization and growth. 44 + 79 pp. Thesis, Swedish University of Agricultural Sciences, Uppsala (1990). ToPA, M.A. and K. W. McLEoD: Responses of Pinus clausa, Pinus serotina and Pinus taeda seedlings to anaerobic solution culture. I. Changes in growth and root morphology. Physiol. Plantarum 68, 523-531 (1986a). - - Aerenchyma and lenticel formation in pine seedlings: A possible avoidance mechanism to anaerobic growth conditions. Physiol. Plantarum 68, 540-550 (1986 b). WUTz, A.: Anatomische Untersuchungen iiber System und periodische Veranderungen der Lenticellen. Bot. Studien 4, 43 -72 (1955).
Occurrence of Lenticels in Roots of Scots Pine Seedlings in Different Growth Conditions TUIJA
S. ARONEN and HELY M. HXGGMAN
The Finnish Forest Research Institute, Punkaharju Research Station, SF-58450 Punkaharju, Finland Received August 17, 1993 · Accepted October 20, 1993
Summary Lenticel tissues were observed in the roots of Scots pine seedlings at different oxygen poor growth conditions: hydroponic culture, in vitro culture, peatland and potted seedlings in the greenhouse. In the roots of seedlings from the typical heath forest site, lenticel formation could not be found. Anatomically, the lenticels consisted of filling cells above the expanded phelloderm region. In peatland seedlings, closing layers were also detectable. The lenticels might be beneficial to the Scots pine at wet growth habitats for gas exchange, even though their proliferation is too slow to be advantageous in flooding incidents.
Key words: Pinus sylvestris, Scots pine, lenticel, roots. Introduction Lenticels are structurally differentiated portions of the periderm characterized by a relatively loose arrangement of cells with intercellular spaces. They exist on almost all longstanding organs like roots and stems, and they have been found both in gymnosperms and in deciduous tree species (Devaux, 1900; Wutz, 1955; Esau, 1965; Buvat, 1989). Structural characteristics of lenticels, like continuity of intercellular spaces with interior parts of stem or root, were interpreted to indicate their functional role in gas exchange (Esau, 1965; Buvat, 1989). This was confirmed in the case of the deciduous tree, Nyssa sylvatica, (Hook et al., 1971), as well as in several coniferous species, e.g. Pinus contorta (Philipson and Coutts, 1978, 1980), Pinus clausa, P. serotina and P. taeda (Topa and McLeod, 1986 b). In hydroponic P. seratina and P. taeda seedlings, stem and root collar lenticels were the major sites of atmospheric 0 2 entry for roots (Topa and McLeod, 1986 b). Scots pine, the economically most important tree species in Finland, is light demanding and grows in different soil types throughout the whole country (Hannelius et al., 1989). The root system and root morphology (Laitakari, 1927) as well as several aspects of mycorrhizal development (e.g. Laiho, 1965; Stenstrom, 1990; Sarjala, 1991) in Scots pine, Pinus sylvestris, have been studied extensively. The existence of lenticels in Scots pine roots has, however, not been reported © 1994 by Gustav Fischer Verlag, Stuttgart
so far. In the present study the development and structure of lenticel tissues have been observed in roots of Scots pine seedlings. The effect of different growth conditions and seed~ing .age on lenticel formation has been studied in vitro and tn
vwo.
Material and Methods Scots pine (Pinus sylvestris L.) seedlings used in the present study were 1) collected from natural stands, 2} potted seedlings in the greenhouse, 3) hydroponic seedlings, and 4) seedlings grown in vitro. Scots pine seedlings were collected from natural stands in Punkaharju (61 o 48' N; 29° 17' E) and in Mekrijarvi (62° 47' N; 30° 57' E). In Punkaharju, 1- to 5-year-old seedlings (n = 29) were collected from the Vaccinium type (VT) site belonging to the dry and dryish heath forest class (Cajander, 1949), where Scots pine stands grow well and are of high quality. In Mekrijarvi, 3- to 10year-old seedlings (n • 26) were collected from a very wet site belonging to the pine peat-moor class (Cajander, 1949), where Scots pines are stunted and of low productivity. The seedlings were spaded with the soil surrounding the roots. In the laboratory the soil was carefully removed under tap water. One- and two-year-old potted seedlings were established with seeds from a local (61 ° 48' N; 29° 17' E) natural stand. The 1-yearold seedlings (n = 50) were grown in containers 8 em in diameter and the 2-year-old ones (n = 50) in containers 13 em in diameter.
326
TUIJA S. ARoNEN and Hm.Y M. HAGGMAN
The hydroponic seedlings (n - 44) were established with the same local seed as the potted ones. They were grown in pots for 4 months and then transplanted into pure solution culture. The seedlings were grown in plastic boxes in the greenhouse, seven seedlings in each 8-L box. The culture solution, replaced completely once a week, included 0.06% commercial fertilizer 5-Superex (Kekkila) containing 10.9% N, 4.0% P, 25.3% K and 1.5% Mg. The solution was aerated by using pressurized air 0.1 Llmin per 1L of culture solution. The 1-year-old in vitro seedlings (n = 378) were established with the local forest seed collected from a natural stand in Punkaharju (61 ° 48' N; 29° 17' E). The surface sterilized (3% Ca-hypochloride) seeds were sown and seedlings grown on ~ GD-media (Gresshoff and Doy, 1972), solidified with 1% agar and without any growth regulators in glass jars 4.5 em in diameter. The seedlings were kept at 23 °C in a 16: 8 h light-dark cycle. The seedlings, with the rest of the old media still surrounding the roots, were transferred into the new jars with fresh media every second month. Occurrence of lenticels in the roots of the seedlings was examined both visually and by using a dissecting microscope. The anatomical structure of lenticels was studied by using a research microscope. Roots with lenticel tissues were fixed in FAA (formalin:asetic acid:95% ethanol, 10:5:85, v/v/v), embedded in paraffm and stained with safranin-fast green according to Gerlach (1984). Surface structures of lenticels were studied by using a scanning electrone microscope (SEM) JEOL JSM-820. The glutaraldehyde (6.25 %)-fixed material was dehydrated in an alcohol gradient, dried by the Critical Point method, mounted on SEM adapters with double-sided adhesive tape and coated with gold.
...
Results and Discussion The different growth conditions remarkably affected the growth habit of the Scots pine seedlings and their roots {Fig. 1). At the typical heath forest site the seedlings had a strong taproot with several lateral roots {Fig. 1 D, 1 E, 1 F). The roots of the peatland seedlings were short and poorly branched. Both the hydroponic {Fig. 1 B) and the potted seedlings {Fig. 1 C) developed large and well branched root systems with taproots. In all of these seedling types the roots were covered with well developed cork. The in vitro seedlings {Fig. 1 A) had no distinguishable taproot, but did have a few slender roots. These roots were covered mostly by epidermis, which in older parts was replaced by thin cork. Lenticel tissues were observed in the roots of Scots pine in different growth conditions: hydroponic culture, in vitro culture, peatland and potted seedlings in the greenhouse. In the roots of seedlings from the typical heath forest site lenticel formation could not be found. In the roots of the potted seedlings the lenticels were small, hardly visible when compared with the size of the lenticels in the seedlings from hydroponic culture, in vitro culture and peatland {Fig. 2). The powdery, pale yellow lenticel tissue appeared as patches protruding from one to several millimeters from the root surface (Fig. 3). In several cases the lenticels were located either beside or around {Fig. 2 D) the emerging lateral roots. The appearance and the amount of the lenticels in Scots pine roots varied according to the growth conditions (Table 1). In the hydroponic seedlings the first lenticels could be observed after 1 month of culture, and after 2 months they
Fig.1: Different root types of Scots pine seedlings developed under different growth conditions. A. In vitro seedling. B. Seedling in hydroponic culture. C. Potted seedling in the greenhouse. D., E., and F. Two, three, and five-year-old heath forest site seedlings, respectively.
could be found in 42% of the seedlings. In in vitro seedlings the first observations of the developing lenticels were made after 2 months of culture. In the potted seedlings, on the other hand, remarkable amounts of lenticels did not appear until the age of 2 years. In the 3- to 10-year-old peatland seedlings, considerable amounts of the lenticels were also detectable. Unfortunately, very young seedlings could not be found at the particular peatland site. The highest amount of
Lenticels in pine roots
,......
..
·~·
t
r
~-------
• :L ··: .:·.::::::-~-:~----·-=--.-.. -..... - :· ·- ····-··----.....- - - -
327
.
,~
'if., ,.;J&t· ~
...
.,..
. . -.-.. -.. .c.-. . . -.. .,. .
-~----
;-.o .........-~--·---·-- •. ,
./1.·J
Fig. 2: Proliferation of lenticels in the roots of Scots pine seedlings. A. In vitro seedling growing on tissue culture media. B. Potted seedling in the greenhouse. Lenticel (arrow) formation in the taproot. C. Seedling in hydroponic culture with remarkable lenticel formation. D. Seedling growing at a very wet site in peatland. Lenticel around the emerging lateral root. Scale bars 3 mm.
Table 1: Occurrence of lenticels in roots of different types of Scots pine seedlings. + = low amount of lenticels; + + = moderate amount of lenticels; + + + = high amount of lenticels. Seedling type
Seedling age (years)
in vitro
n- 378
Fig. 3: Scanning electron micrograph presenting the surface structure of a lenticel in the roots of a Scots pine in vitro seedling.
Occurrence of lenticels in roots of seedlings studied
Amount of lenticels per seedling
54
++
(%)
hydroponic n = 44 n- 44
2 months 1 7 months 1
42 95
+++ +++
potted n =50 n • 50
1 2
4 44
+ ++
heath forest n • 11 n- 14 n=4
1-2 3 4-5
0 0 0
peatland n • 26
3-10
46
1
Months in hydroponic culture.
++
328
TUJJA S. ARoNEN and Hm.Y M. HXGGMAN
lenticels per seedling was found in hydroponic culture, where the roots were almost covered with lenticel tissues. In in vitro, peatland and 2-year-old potted seedlings the number of lenticels per seedling varied from a few single ones up to several dozens. In 1-year-old potted seedlings the lenticel number was lowest, only one or two per seedling. All of the growth conditions, inducing lenticel formation, could be considered as oxygen poor environments. In typical peatland sites, according to Hannelius and co-workers (1989), Scots pine seedlings suffer from deficiency of air space together with minor oxygen content in soil. Hydroponic culture systems have also earlier been used to imitate anaerobic environments (Topa and McLeod, 1986 a). The in vitro grown Scots pine seedlings have been observed to grow better with a continuous supply of air (Flygh et al., 1988). In potted seedlings the formation of smalllenticels after 2 years could be due to the shortage of oxygen caused by the excess of roots in a limited space (plastic pot). The lenticels were abundant in old cork covered roots of all seedling types, but they were also recognizable in young organs, like in epidermis covered roots, especially in hydro-
ponic and in vitro seedlings. This phenomenon was already described by Esau (1965) and Buvat (1989). Topa and McLeod (1986a) reported remarkable lenticel formation in pond and loblolly pines after 30 days of hydroponic culture, but in the case of sand pine the lenticel formation was nearly absent. According to our results considerable amounts of lenticels in Scots pine appeared much later than 1 month. Thus, the appearance of lenticels in Scots pine resembles more that of sand pine than that of pond or loblolly pines. Anatomically, lenticel tissues in Scots pine roots consisted of filling cells arising from repited cell divisions in phellogen. Filling cells were of angular form, more or less elongated (Fig. 4 B, 4 C). Intercellular air spaces between filling cells expanded towards the surface of the lenticels (Fig. 4 A, 4 B). Closing layers in lenticels were found only in the roots of the peatland seedlings (Fig. 4 B). In the lenticel regions phelloderm was usually expanded, consisting of several layers of compactly arranged cells (Fig. 4 A, 4 C). This was particularly true in the case of in vitro seedlings, in which the lenticels had only a limited number of the filling cells (Fig. 4 A). Throughout the pericycle region intercellular air
..
·"'"'
··~
...
..
. . ~ ,, .
,·'
· ..
..
Fig. 4: Anatomical characteristics of lenticels in the roots of Scots pine seedlings. A. Anatomical structure of a lenticel on the root of an in vitro seedling. B. A lenticel of a peatland seedling with characteristic closing cell layers and abundant amounts of filling cells. C. Expanded phelloderm region in the lenticel of an in vitro seedling. D. Intercellular air spaces throughout the pericycle region of an in vitro seedling. Scale bar in A. and B. 500 J.lm and in C. and D. 100 J.lm. Abbreviations: CL, closing layer; CW, cell wall fragments; FC, filling cells; IS, intercellular air spaces; Pc, pericycle; Pd, phelloderm; Pg, phellogen; X, xylem.
Lenticels in pine roots
spaces were abundant, extending from the phelloderm to the phloem {Fig. 4D). The cells of the pericycle typically contained a lot of starch grains {Fig. 4 D). The air spaces in the pericycle appeared to be predominantly lysigenous due to cell wall fragments present in aerenchyma. Lenticels in the roots of conifers have been studied by Topa and McLeod ( 1986 a, 1986 b). They could not find any closing cells in lenticels of pond and loblolly pines, or expanded phelloderm regions under the filling cells. They also reported that the intercellular air spaces in the pericycle of anaerobically grown pine seedlings were particularly prominent adjacent to a lenticel. These anatomical differences between the results of the present study and those of T opa and McLeod's could be due to different durations of the oxygenpoor growth conditions, different age of the seedlings, or different pine species. In other respects the anatomical structure of Scots pine lenticels corresponded with that described earlier in pond and loblolly pine (Topa and McLeod, 1986 b). Expanded phelloderm regions, such as we found in Scots pine, have also been reported with other species like Coriaria myrtifolia (Devaux, 1900). Lenticels with closing layers have been described in many decidious tree species already by Devaux (1900), but until the present study they have not been found in coniferous species. The abundance of intercellular air spaces in the Scots pine roots could indicate adaptation to anaerobic growth conditions as described earlier in angiosperms by Davy and co-workers (1990), Jackson (1990) and Peterson {1992). According to the present results the oxygen poor growth conditions cause anatomical adaptations in Scots pine roots. Previous investigations have revealed that the ability of different pine species to form lenticels correlates to their flooding tolerance, being abundant in flooding tolerant species (Topa and McLeod, 1986a). Our results show that the flooding intolerant .Scots pine (Hannelius et al., 1989) is able to form remarkable amounts of lenticels. The lenticel proliferation in Scots pine roots, however, seems to take too long to be beneficial in flooding incidents. Although Scots pine can not survive flooding, it is common in wet growth habitats like in peatlands (Paavilainen and Tiihonen, 1988), where the lenticels might be advantageous. Acknowledgements We are grateful to Dr. Anneli Kauppi for critical reading of the manuscript. We would also like to thank Ms. Rauni Ritola, Ms. Rauni Kantola and Mr. Jouko Lehto for technical assistance during the work.
References BuvAT, R.: Ontogeny, cell differentiation and structure of vascular plants, 2nd ed. Springer-Verlag, Berlin, Heidelberg (1989).
329
CAJANDEll, A. K.: Forest types and their significance. Acta For. Fenn. 56, 1-71 (1949). DAVY, A. J., S.M. NoBLE, and R. P. OuVEll: Genetic variation and adaptation to flooding in plants. Aquat. Bot. 38, 91-108 (1990). DEvAux, H.: Recherches sur les lenticelles. Ann. des Sci. Nat., Bot. Ser. 8. 12, 1-240 (1900). ESAu, K.: Plant Anatomy, 2nd ed. John Wiley & Sons, New York (1965). FLYGH, G., G. GllONRoos, and S. voN AllNoLD: Optimizing growth of conifer seedlings in vitro. Physiol. Plant. 73 (2), 20 A (1988). GEllLAcH, D.: Botanische Mikrotechnik. George Thieme Verlag, Stuttgart (1984). GllESSHOFF, P. M. and C. DoY: Development and differentiation of haploid Lycopersicon esculentum (Tomato). Planta 107, 161-170 (1972). HANNE:uus, S., M. LmKoLA, and A. TUIMALA: Metsakirja, metsanomistajan kasikirja. WSOY, Porvoo (1989). HooK, D. D., C. L. BlloWN, and P. P. KollMANIK: Inductive flood tolerance in swamp tupelo (Nyssa sylvatica var. biflora (Walt.) Sarg.).J. Exp. Bot. 22,78-89 (1971). jACKSON, M. B.: Hormones and developmental change in plants subjected to submergence or soil waterlogging. Aquat. Bot. 38, 4972 (1990). LAnio, 0.: Further studies on the ectendotrophic mycorrhiza. Acta For. Fenn. 79, 1-35 (1965). LArrAKAlll, E.: The root system of pine (Pinus sylvestris). A morphological investigation. Acta For. Fenn. 33, 307-380 (1927}. PAAVILAINEN, E. and P. TITHONEN: Peatland forests in Finland in 1951-1984. Folia Forestalia 714, 1-29 (1988). PETEllSON, R. L.: Adaptations of root structure in relation to biotic and abiotic factors. Can. J. Bot. 70, 661-675 (1992). PHIUPSON, J. J. and M.P. CouTTs: The tolerance of tree roots to waterlogging. III. Oxygen transport in lodgepole pine and Sitka spruce roots of primary structure. New Phytol. 80, 341-349 (1978). - - The tolerance of tree roots to waterlogging. IV. Oxygen transport in lodgepole pine and Sitka spruce roots of primary structure. New Phytol. 85, 489-494 (1980). SARJALA, T.: Nitrogen assimilation in ectomycorrhizal fungi and mycorrhizal and non-mycorrhizal Scots pine seedlings. Acta Univ. Oul. A218 (1991}. STENsnOM, E.: Ecology of mycorrhizal Pinus sylvestris seedlings aspects of colonization and growth. 44 + 79 pp. Thesis, Swedish University of Agricultural Sciences, Uppsala (1990). ToPA, M.A. and K. W. McLEoD: Responses of Pinus clausa, Pinus serotina and Pinus taeda seedlings to anaerobic solution culture. I. Changes in growth and root morphology. Physiol. Plantarum 68, 523-531 (1986a). - - Aerenchyma and lenticel formation in pine seedlings: A possible avoidance mechanism to anaerobic growth conditions. Physiol. Plantarum 68, 540-550 (1986 b). WUTz, A.: Anatomische Untersuchungen iiber System und periodische Veranderungen der Lenticellen. Bot. Studien 4, 43 -72 (1955).