Botanical Journal of the Linnean Society, 2008, 158, 548–555. With 16 figures Formation of successive cambia and stem anatomy of Sesuvium sesuvioides (Aizoaceae) KISHORE S. RAJPUT*, VIDYA S. PATIL and DHARMENDRA G. SHAH Received 15 February 2008; accepted for publication 14 April 2008 Mature stems of Sesuvium sesuvioides (Fenzl) Verdc. were found to be composed of successive rings of xylem alternating with phloem. Repeated periclinal divisions in the parenchyma outside the primary phloem gave rise to conjunctive tissue and the lateral meristem that differentiate into the vascular cambium on its inner side. After the formation of the vascular cambium, the lateral meristem external to it became indistinct as long as the cambium was functional. As the cambium ceased to divide, the lateral meristem again became apparent prior to the initiation of the next cambial ring. The cambium was exclusively composed of fusiform cambial cells with no rays. In the young saplings, the number of cambial cylinders in the axis varied from the apex to the base, indicating formation of several rings within the year. In each successive ring of the lateral meristem, small segments differentiated into the vascular cambium and gave rise to vessels, axial parenchyma, fibres and fibriform vessels towards the inside, and secondary phloem on the outer side. In the old stems, non-functional phloem of the innermost rings was replaced by a new set of sieve tube elements formed by periclinal divisions in the cambial segments associated with the non-functional phloem. In some places the cambial segments completely differentiate into derivatives leaving no cambial cells between the xylem and phloem. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 548–555. ADDITIONAL KEYWORDS: cambial variant – nucleated xylem fibres – raylessness – secondary cortex. INTRODUCTION The phenomenon of successive cambia in Aizoaceae has been known for many years and is considered to be a characteristic feature of the family (Pax & Hoffman, 1934; Rao & Rajput, 1998; Carlquist, 2007a, b). Although to varying degrees Aizoaceae are characterized as a succulent, members are found in a wide range of habitats. In the present study, plants growing on sand and in rock crevices at the seashore were collected. Owing to the presence of successive cambia, different members of Aizoaceae have been studied from time to time by various workers (Schenck, 1893; Pfeiffer, 1926; Metcalfe & Chalk, 1950, 1983; Rao & Rajput, 1998; Carlquist, 2007a), but there is no detailed information on the pattern of secondary growth and the structure of the secondary *Corresponding author. E-mail: ksrajput@yahoo.com 548 xylem in Sesuvium sesuvioides (Fenzl) Verdc. (Aizoaceae). It has been considered that, during the course of evolution, different groups of plants have undergone various modifications, which may be biochemical, morphological or structural. These modifications helped the plants to adapt to particular climatic or ecological conditions. Among these were structural modifications; especially that pattern of secondary thickening remains characteristic of a specific group. These patterns of secondary thickening include formation of successive cambia, rayless xylem and paedomorphosis, and the formation of included phloem or of internal phloem etc. These features are well documented in most of the families of core Caryophyllales with perennial stems. Formation of successive cambia has also been reported in more than 20 families outside core Caryophyllales (Carlquist, 2007a: 1), as well as in some gymnosperms such as Gnetum, Welwitschia (Carlquist, 1996), Cycas revoluta (Handa, © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 548–555 Downloaded from https://academic.oup.com/botlinnean/article-abstract/158/3/548/2418344 by guest on 28 May 2020 Department of Botany, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara–390 002, India CAMBIA AND STEM ANATOMY OF S. SESUVIOIDES RESULTS STRUCTURE OF THE CAMBIUM Mature stems of S. sesuvioides (Fenzl) Verdc. were composed of three to five successive rings of cambia (Figs 1, 2) comprised entirely of fusiform cambial initials with no cambial rays (Fig. 3). The cambium was semi-storied with relatively short fusiform cambial cells varying from 217–229 mm in length and 17–23 mm in width. In the transverse view, the cambium appeared two- to three-layered when nondividing and four- to six-layered during the development of the xylem and phloem. In each successive ring of lateral meristem, small segments differentiated into the vascular cambium, giving rise to the conducting elements of xylem and phloem in the centripetal and centrifugal directions, respectively (Figs 2, 4). These small segments of cambium were interconnected by wider segments of lateral meristem, which produced only thick-walled conjunctive tissue centripetally and thin-walled parenchyma (Figs 2, 4) centrifugally. The structure of the cambium and xylem in the stem and roots remained more or less similar with the exception of the parenchymatous conjunctive tissue, which was more common in the roots compared with the stem. MATERIAL AND METHODS From the plants of S. sesuvioides, 8–10 segments of the main stems and roots measuring 3–20 mm in diameter and 40–60 mm in length were collected. These plants were growing on the sea coast of Gujarat State. The stem segments adjacent to ground level and the roots of ten plants were immediately fixed in formalin–acetic acid–alcohol (FAA) (Berlyn & Miksche, 1976). After suitable trimming, these segments were dehydrated in tertiary butyl alcohol (TBA) series and processed using the routine method of paraffin embedding. Transverse, radial and tangential longitudinal sections of 12- to 15-mm thick were microtomed using either a Leica rotary microtome and stained with safranin fast green combination (Johansen, 1940) or a tannic acid–ferric chloride– lacmoid combination (Cheadle, Gifford & Esau, 1953). Thick stem segments were also sectioned on a sliding microtome. The length of the fusiform cambial cells was calculated directly from the tangential longitudinal sections. Pieces of the outermost xylem adjacent to the cambium of approximately 1-mm2 thick were macerated using Jeffrey’s solution (Berlyn & Miksche, 1976) at 55–60 °C for 24–36 h to study the general morphology and size of the vessel elements and fibres. One hundred measurements were chosen randomly from each specimen to calculate the mean and standard deviation. Important results were microphotographed using a photomicroscope. DEVELOPMENT OF THE VASCULAR CAMBIUM In the young stem, several collateral vascular bundles were joined by interfascicular cambium to form a complete cylinder of vascular cambium. The first ring of cambium ceased to divide after a limited period of activity and the second ring of cambium developed from the axial parenchyma cells at a distance of about three to six cell layers outside the phloem produced by the previous cambium (Figs 4–7). Prior to the initiation of the second ring, these parenchyma cells underwent repeated periclinal divisions to form a wide band of cells (referred to here as the secondary cortex) that served as a site for the origin of the lateral meristem (Fig. 7). Cells of the secondary cortex were relatively smaller in diameter compared with the cortical parenchyma. However, the cells located in the middle of the secondary cortex divided repeatedly to form a radially arranged lateral meristem two to three cells wide (Figs 6, 7). Four to five layers of parenchyma between the newly developed cambium and the phloem produced by the previous cambium differentiated into conjunctive tissues, while cells located externally to the new cambium divided further, giving rise to the secondary cortex. The lateral meristem became more conspicuous when a new ring of cambium was to be developed and it became quiescent when the vascular cambium internal to it became active. A small alternating segment © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 548–555 Downloaded from https://academic.oup.com/botlinnean/article-abstract/158/3/548/2418344 by guest on 28 May 2020 1939) and Encephalartos, Lepidozamia and Macrozamia (Stevenson, 1990). A characteristic feature of core Caryophyllales is the formation of successive cambia. Variations in the cambial products, such as more parenchymatous conjunctive tissue and less thick walled lignified elements, or vice versa, have always posed problems to different workers. Thus, successive cambia in this group represent a mode of structure that has been variously interpreted with respect to its origin, nature and products (Carlquist, 2007a: 141). It has also been found that the terminology applied to stem and root histology in species with successive cambia is as diverse as the interpretations and it seems certain that each family with successive cambia could potentially offer important information on this phenomenon (Carlquist, 2007a: 141). However, the nature of the products requires features such as habit and texture to be correlated for a better understanding of this phenomenon. Therefore, in the present study, an attempt was made to correlate the pattern of secondary growth with the habit of the plants and to elucidate the structure of the xylem and the development of the cambial variant in S. sesuvioides. 549 550 K. S. RAJPUT ET AL. of the lateral meristem differentiated into conducting elements of xylem and phloem, while the remaining segments gave rise to thick-walled conjunctive tissue centripetally and thin-walled parenchymatous conjunctive tissue centrifugally. The formation of subsequent cambia followed a similar pattern of development. STRUCTURE AND DEVELOPMENT OF VASCULAR ELEMENTS In mature stems, the xylem was composed of vessels, tracheids, fibriform vessels and fibres, while xylem rays were found to be absent. Xylem fibres, as well as thick-walled conjunctive tissue formed towards the © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 548–555 Downloaded from https://academic.oup.com/botlinnean/article-abstract/158/3/548/2418344 by guest on 28 May 2020 Figures 1–4. Transverse (Figs 1–2, 4) and tangential longitudinal (Fig. 3) view of the stem of Sesuvium sesuvioides. Fig. 1. Young stem showing arrangement of vascular tissues from epidermis to secondary xylem. Note the variation in the cell size of the cortical parenchyma and the secondary cortex. Fig. 2. Mature stem with three successive rings of xylem alternating with phloem. Note that the formation of vessels and phloem remains restricted to certain segments of the cambium. Fig. 3. The semi-storied nature of the cambium showing an absence of rays. Fig. 4. Newly developing cambium. The arrowheads indicate the origin of the lateral meristem from the parenchyma cells outside the phloem produced by the previous cambium. Note the variation in the cell size of the cortical parenchyma and the secondary cortex (arrow). CAMBIA AND STEM ANATOMY OF S. SESUVIOIDES 551 xylem side, retained their nuclei even in the innermost xylem rings of the mature stem. As the secondary growth progressed further, the sieve tube elements associated with the innermost rings of xylem became non-functional by heavy accumulation of callose and gradually underwent obliteration (Figs 8–11). This non-functional phloem was replaced by the formation of new sieve tube elements from the cambial cells present between the secondary xylem and phloem (Fig. 10). However, differentiation of new © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 548–555 Downloaded from https://academic.oup.com/botlinnean/article-abstract/158/3/548/2418344 by guest on 28 May 2020 Figures 5–10. Transverse view of stem showing origin of vascular cambium and structure of xylem in Sesuvium sesuvioides. Fig. 5. The newly developing cambium (arrowhead) from the parenchyma cells outside the phloem produced by the previous cambium. Note the relatively larger size of cortical parenchyma than the secondary cortex. Fig. 6. Enlarged view of Figure 5 showing the initiation of the lateral meristem from the parenchyma cells outside the phloem formed by the previous cambium (arrowheads). Fig. 7. The newly developed cambium (arrowheads) with few differentiated xylem derivatives. Fig. 8. Complete differentiation of the cambial region in the innermost ring of the cambium (arrowhead). Note the thick-walled lignified conjunctive tissue (arrow); see also Figure 2. Fig. 9. The obliterated non-functional phloem (arrowhead) is replaced by newly formed phloem. Note the complete differentiation of cambium and conjunctive tissue with lignified walls (arrow). Fig. 10. Cambial region showing newly differentiated sieve tube elements (arrow). Arrowhead indicates crushed phloem. C, cambium. 552 K. S. RAJPUT ET AL. DISCUSSION In S. sesuvioides, the stem increases in thickness by forming successive rings of cambia. These cambia produce secondary xylem centripetally and secondary phloem centrifugally, while lateral meristems form conjunctive tissue on either sides of it. Successive cambia are known to occur in the majority of the genera belonging to Caryophyllales and are well known as a cause of concentric rings of secondary xylem and secondary phloem, which are embedded in a background of parenchyma or fibre groundmass. Plants with successive cambia possess varied growth forms and habitats. Therefore, it becomes difficult to correlate between growth forms, habitats and formation of successive cambia. However, they share a common character, such as a similarity in pattern of secondary growth and the origin of the lateral meristem. Although S. sesuvioides grows as a halophyte, the pattern of secondary growth and the origin of the lateral meristem remain similar to Trianthema monogyna of Aizoaceae (Rao & Rajput, 1998) and the species of Molluginaceae (Rao & Rajput, 2003). Several collateral vascular bundles were joined by the interfascicular cambium to form a complete ring of vascular cambium. In this ring, the differentiation of conducting elements of xylem and phloem remained restricted to the fascicular segments. In contrast, the interfascicular cambium produced only thick-walled conjunctive tissue centripetally and thinwalled cells centrifugally. After a definite period of activity it becomes non-functional by cessation of cell division. Before it ceased to divide, parenchyma cells outside the phloem underwent repeated periclinal divisions to form a secondary cortex. This secondary cortex acted as a site for the origin of a new meristem in the future. A similar pattern of cambial origin was reported previously in Amaranthaceae (Rajput & Rao, 2000; Rajput, 2001, 2002; Carlquist, 2003), Chenopodiaceae (Fahn & Zimmermann, 1982), Nyctaginaceae (Rajput & Rao, 1998; Carlquist, 2004), Trianthema monogyna (Rao & Rajput, 1998) and other members of Aizoaceae (Carlquist, 2007a, b). Stems and roots of Aizoaceae develop a lateral meristem within the cortical parenchyma after maturation of the primary vasculature. This meristem produces a secondary cortex to the outside and conjunctive tissue and vascular cambia to the inside. Carlquist (2004) proposed a new term for such a meristem: the ‘Lateral meristem’. According to him, vascular cambia exist only where secondary phloem is produced to the outside and vessels (with or without production of fibres) to the inside of vascular cambium. However, lateral meristem produce only conjunctive tissue, while vessels to the inside and secondary phloem to the outside are absent. Conjunctive tissue formed by lateral meristem may have thin or thick walls and cells may be fibre-like or parenchymatous, with every transition between the two types (Carlquist, 2004). All these are characteristic © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 548–555 Downloaded from https://academic.oup.com/botlinnean/article-abstract/158/3/548/2418344 by guest on 28 May 2020 sieve tube elements from the cambial cells exerted a pressure on the non-functional phloem that ultimately got crushed (Figs 8–10). In mature stems, some of the innermost cambial segments completely differentiated into sieve tube elements and parenchyma cells leaving no cambial cells between the secondary xylem and phloem (Fig. 8). At such places where the cambial segments were completely differentiated into their derivatives, the thin-walled conjunctive tissue around them also underwent lignification and became thick walled (Figs 8, 9). As mentioned earlier, the development of conducting elements of xylem and phloem was confined to certain portions of the cambial segments. This led to the development of vessels mostly in radial files. They had simple perforation plates with alternate bordered pits on their lateral walls. The average length and width of vessel elements was measured to be approximately 159–195 mm and 48–78 mm, respectively, while fibriform vessels were very narrow and indistinguishable in the transverse view. They measured approximately 210–218 mm in length and 23–28 mm in width. Xylem fibres retained their living protoplast and nuclei even after the deposition of the secondary wall material. The nuclei were oval to oblong or elongated to fusiform in shape (Figs 12, 13). The length of nuclei, including the sharp points, varied from 5 to 10.5 mm. Morphologically, the xylem fibres varied greatly: some of the fibres were straight and spindle shaped while others had undulating walls. Although uncommon, the occurrence of branched fibres was observed frequently. Pits on the walls were simple and confined to radial walls. Their slit-like aperture formed a narrow angle with the fibre axis. The length of fibres varied from 442–489 mm. In the initial phase of secondary growth, the xylem possessed very few vessels with a larger diameter and was mainly composed of fibriform vessels. From the second ring of the cambium onwards, the xylem was by and large composed of wider vessels with very few fibriform vessels (Fig. 14). Similarly, in the first two successive rings of xylem, the conjunctive tissues formed to the inside were mostly thick walled, but, as the number of cambial rings increased, formation of thick-walled conducting elements remained restricted only to the small segments of cambium, while the longer alternate segment of it formed thin walled parenchymatous conjunctive tissue on either side of the cambium. Like the fusiform cambial cells, the xylem fibres, tracheids and axial parenchyma were also arranged in storied fashion (Figs 15, 16). CAMBIA AND STEM ANATOMY OF S. SESUVIOIDES 553 features of S. sesuvioides and are in agreement with characters proposed for the lateral meristem by Carlquist (2004). If this feature of a lateral meristem is correlated with fascicular and interfascicular regions of the first ring of cambium, formation of conducting elements of secondary xylem and phloem remains restricted to the fascicular region of the cambium, while formation of conjunctive tissue from the lateral meristem coincides with the interfascicular region. Such cambia are also reported to be © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 548–555 Downloaded from https://academic.oup.com/botlinnean/article-abstract/158/3/548/2418344 by guest on 28 May 2020 Figures 11–16. Tangential (Figs 13, 15), transverse (Figs 11, 12, 14) and radial longitudinal (Fig. 16) views of the secondary xylem in Sesuvium sesuvioides. Fig. 11. Prior to the obliteration, sieve tube elements show heavy accumulation of callose (arrowhead). Fig. 12. Xylem fibre with a nucleus (arrow). Arrowheads indicate a thick wall with pit aperture. Fig. 13. Xylem fibre with a nucleus (arrows). Fig. 14. Structure of xylem in first two successive rings. Note that the narrow vessels are more in the first ring (lower half) while the second ring is predominated with wider vessels. Fig. 15. The structure of the xylem showing the absence of rays. Fig. 16. Structure of the xylem. 554 K. S. RAJPUT ET AL. stems, non-functional phloem of the inner rings was replaced by the addition of new sieve tube elements by cell division activity of the vascular cambium. This cambium produced only sieve tube elements and no xylem elements because there was no scope for the enlargement of differentiating xylem elements. However, secondary phloem could be added because earlier formed phloem was crushed as new phloem was added. To what extent does this crushed phloem accumulate in the innermost successive rings? Does the cambium produce secondary phloem for an indefinite period? Does the cambium of the innermost successive rings maintain a radial arrangement? No information is available on these aspects, which have always been neglected. It may be as a result of the unavailability of wet preserved material and the fact that most of the studies on cambial variants are based on the material available from the xyleria. In the present study, in most of the innermost rings, the vascular cambium completely differentiated into its derivatives. At these places, the radial arrangement of cambial cells is lost, while surrounding thin-walled conjunctive tissue becomes thick walled by deposition of lignin (Figs 8, 9). Here, it seems that, after a definite period of activity, the cambium in S. sesuvioides loses its cell division ability, thus differentiating into its derivatives. This statement, however, cannot be generalized to all the plants showing successive cambia and needs to be confirmed for other plants. For what duration are these cambia able to differentiate into phloem and maintain their radial arrangement? A similar condition has also been reported in Boerhaavia in an earlier study (Rajput & Rao, 1998: 132). The occurrence of nucleated xylem fibres seems to be associated with the rayless nature of xylem. It is considered to be an adaptive feature, associated with the diminishing supportive function, exhibiting a transition towards parenchyma cells that prevails in herbaceous plants (Fahn & Leshem, 1963). Moreover, accumulation of starch in these fibres also suggests that, in addition to mechanical support, they also act as a reservoir of photosynthetic products representing a further functional connection between parenchyma cells and wood fibres (Rajput & Rao, 1999b, c). Parmeswaran & Liese (1969) correlated the ability of xylem fibres to accumulate starch with a small number of storage parenchyma. The presence of nucleated xylem fibres suggests that these fibres may not only act as a reservoir of photosynthate but also play an important role in radial conduction. Thus, in S. sesuvioides, the presence of nucleated fibres represents a transition between the parenchyma and the fibres, which may be performing a dual function as mechanical tissue as well as storage cells in the absence of rays. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 548–555 Downloaded from https://academic.oup.com/botlinnean/article-abstract/158/3/548/2418344 by guest on 28 May 2020 rayless most of the time, which is also true in the case of S. sesuvioides. Raylessness tends to occur in those groups of plants in which cambial activity is limited, thus resulting in scanty accumulation of the secondary xylem (Carlquist, 1970). Sometimes this may be seen in plants with considerably thicker stems, as observed in case of Bougainvillea. If a single cambium can no longer produce xylem, then successive cambia may increase stem or root diameter. Such a xylem is always rayless (Barghoorn, 1941; Carlquist, 1970; Rajput & Rao, 1998; Rao & Rajput, 1998; Rajput & Rao, 1999a). However, S. sesuvioides shows scanty accumulation of secondary xylem by the formation of successive cambia in thick stems. Absence of rays is not a common feature and is restricted to a small section of dicotyledons belonging to quite different families (Carlquist, 1988; Lev-Yadun & Aloni, 1995; Rao & Rajput, 1998; Rajput & Rao, 1998, 1999a). In Caryophyllales, raylessness occurs in a number of species that show successive cambia. In Aizoaceae, all the species studied so far are reported to be rayless, with the exception of Tetragonia (Carlquist, 2007a). In S. sesuvioides, the secondary xylem remains rayless and the stem increases in thickness by forming successive cambia. Our observation on the origin of the lateral meristem is in agreement with Carlquist (2007a): that it first originated in the cortex of the primary stem. However, vascular cambia develops from the lateral meristem on its inner face and it is not used up in giving rise to the vascular cambium (Carlquist, 2004: 141). When one cylinder of cambium ceases to divide, a quiescent lateral meristem appears outside the vascular cambium leaving a segment of cambium sandwiched between the secondary xylem and phloem. As reported by Carlquist (2004: 129), quiescence of the lateral meristem at these points coordinates the amount of tissue produced to the inside in zones without vascular cambia with that produced in zones with cambia. It is a fact that successive cambia have no relationship to annual events and the known chronological age of the plants (Carlquist, 2007a: 151). In the present study, a young sapling of less than 1 year was sectioned at different points, starting from the tip and moving towards the main stem to check the number of cambial cylinders. 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