IAWA Journal, Vol. 32 (4), 2011: 475–491 DEVELOPMENT OF INTRA- AND INTERXYLARY SECONDARY PHLOEM IN COCCINIA INDICA (CUCURBITACEAE) Vidya S. Patil1, Carmen R. Marcati2 and Kishore S. Rajput1,* SUMMARY Stem anatomy and the development of intraxylary phloem were investi- gated in six to eight years old Coccinia indica L. (Cucurbitaceae). Sec- ondary growth in the stems was achieved by the normal cambial activity. In the innermost part of the thicker stems, xylem parenchyma and pith cells dedifferentiated into meristematic cells at several points. In some of the wider rays, ray cells dedifferentiate and produce secondary xylem and phloem with different orientations and sometimes a complete bicol- lateral vascular bundle. The inner cambial segments of the bicollateral vascular bundle (of primary growth) maintained radial arrangement even in the mature stems but in most places the cambia were either inactive or showed very few cell divisions. Concomitant with the obliteration and collapse of inner phloem (of bicollateral vascular bundles), pa- renchyma cells encircling the phloem became meristematic forming a circular sheath of internal cambia. These internal cambia produce only intraxylary secondary phloem centripetally and do not produce any sec- ondary xylem. In the stem, secondary xylem consisted mainly of axial parenchyma, small strands of thick-walled xylem derivatives, i.e. ves- sel elements and fibres embedded in parenchymatous ground mass, wide and tall rays along with exceptionally wide vessels characteristic of lianas. In thick stems, the axial parenchyma de-differentiated into meristem, which later re-differentiated into interxylary phloem. Fibre dimorphism and pseudo-vestured pits in the vessels are also reported. Key words: Coccinia indica, interxylary meristem, bicollateral vascular bundles, ray cambium. INTRODUCTION Climbing plants differ from trees and shrubs in a number of characteristics; most no- table in the mechanical properties of the stem (Isnard et al. 2003; Lopes et al. 2008). The shift from self supporting to the climbing habit co-occurs with the development of structural patterns, which differ from the more usual anatomical types, and are some- times referred as anomalous (Cutter 1969) or cambial variant (Carlquist 1988; Sajo & 1) Department of Botany, Faculty of Science, The M. S. University of Baroda-390002, India. 2) Departamento de Recursos Naturais, Faculdade de Ciências Agronômicas, Universidade Estadual Paulista, UNESP, Campus de Botucatu, SP, Brazil. *) Corresponding author [E-mail: [email protected]]. Associate Editor: Frederic Lens Downloaded from Brill.com09/26/2021 12:42:32PM via free access 476 IAWA Journal, Vol. 32 (4), 2011 Castro 2006), although these variants may also occur in non-climbing shrubs and trees. Different types of cambial variants occur in lianas that are considered to increase stem flexibility, to protect the conducting elements of xylem and phloem, and to increase storage parenchyma. They may also limit physical disruption of vascular tissues during twisting and bending, and promote wound healing after girdling (Dobbins & Fisher 1986; Fisher & Ewers 1992; Lopes et al. 2008); all aspects that are functionally relevant in the lianescent stems. The Cucurbitaceae contain 97 genera and about 940–980 species (Schaefer & Renner 2011), in which most of the genera are non-woody and have thus been neglected from a wood anatomical point of view, but there are a few moderately woody genera as well, some of which have been studied anatomically by Carlquist (1992). The stem anatomy of Dendrosicyos socotrana, the only arborescent member of the family, was examined by Olson (2003) who tried to establish correlations to test hypotheses regarding features adaptive to scandent and erect life forms. In this species, stems were mainly composed of very wide rays, parenchymatous conjunctive tissues like lianas, but wide vessels with thick walls were lacking (Olson 2003). According to him, other features such as very wide rays and presence of abundant axial parenchyma in both trees and lianas appear to serve different roles in both life forms (Dobbins & Fisher 1986; Fisher & Ewers 1992; Carlquist 2001). The small strands of xylem in Dendrosicyos socotrana were associated with segments of subsequent vascular cambia. This constitutes the first report of successive cambia in the Cucurbitales (Olson 2003). Later, successive cambia were also reported in Bryonia and Ecballium (Schweingruber et al. 2010). Coccinia is a moderately woody climber but it shares most of the anatomical features reported by Olson (2003) for Dendrosicyos, but it lacks successive cambia. In Coccinia, stem thickness is achieved by several meristematic bands in rays and other parts of the secondary xylem. The radial arrangement of these cells in the meristematic bands gives them a cambium-like appearance. In India, most of the members of the family are annual herbs, which start growing with the germination of seeds in June with the arrival of rains and complete their life cycle after the dispersal of fruits in January-February. Therefore, in most of the gen- era secondary growth is limited and stem thickness does not exceed 5 cm. In Gujarat state (India), about 15 genera and 31 species of the Cucurbitaceae are reported (Shah 1978). Among them, Coccinia indica is the only species that is perennial and more woody. Therefore it has been selected in the present investigation to study the pattern of secondary growth. In the dry scrub forest of Gujarat state, Coccinia is one of the main climbing species found growing on Acacia spp. The presence of two different types of phloem (fascicular and extrafascicular) in the Cucurbitaceae has been recognized for a very long time (Fischer 1883; Crafts 1932). The ease with which phloem sap can be collected and the presence of a unique type of vascular bundles in some cucurbits, have made them excellent candidates for stud- ies of phloem transport and determining the composition of the sap in the conducting sieve elements (Turgeon & Oparka 2010; Zhang et al. 2010). The fascicular phloem is restricted to either side of the main xylem in the main vascular bundles, whereas extrafascicular phloem forms an anastomosing network that interconnects the vascular Downloaded from Brill.com09/26/2021 12:42:32PM via free access Patil, Marcati & Rajput — Phloem of Coccinia 477 bundles laterally in the stem and petiole (Turgeon & Oparka 2010; Zhang et al. 2010). Functionally, fascicular phloem is associated with the transport of the products of photosynthesis and other nutrients over long distances from the leaves to sink tissue. In contrast, Golecki et al. (1999) and Zhang et al. (2010) reported that extrafascicular phloem is associated with the transport of macromolecules, proteins, abundant amino acids and a wide range of unidentified secondary metabolites while it contains very low level of sugars. In most of the dicots with normal phloem, the seasonal cycle of phloem develop- ment has been well studied (Davis & Evert 1968; Kozlowski 1971; Ghouse & Hashmi 1983; Vishwakarma 1991; Rajput & Rao 1998). In general, every year, older and non- functional phloem is replaced with new phloem by the seasonal activity of the vascular cambium. Although the anatomy of the Cucurbitaceae is well documented in the major- ity of reference books, no information is available on the renewal of inner phloem of bicollateral vascular bundles of mature plants, i.e., plants over 6–8 years old. More- over, the stems of Coccinia are soft due to the abundance of parenchyma cells. What happens to these cells? Do they undergo lignification or are these parenchyma cells subject to collapse and replacement by other cells or cell types? The main aims of the present investigation were: i) to study the structural alterations occurring in the parenchymatous secondary xylem after 6–8 years, ii) to study whether earlier formed phloem is replaced by new located on the inner side of the bicollateral vascular bundles in 6–8-year-old stems, iii) to study the significance of parenchyma abundance in the stem of Coccinia indica, and iv) to put our results in a comparative context of the anatomical range in the Cucurbitaceae. MateRIALS AND METHODS Eight to ten segments measuring about 10–50 mm thick and 40–60 mm in length were collected from the main stems of ten plants of Coccinia indica L. (Cucurbitaceae) growing in the University campus and Arboretum of the Botany Department of the M.S. University of Baroda, India. The plants collected were approximately 6–8 years old. These samples were fixed immediately in FAA (formalin, acetic acid, 70% ethyl alcohol; 10 : 5 : 85, v/v) and after 12 hrs of fixationcut into smaller pieces and transferred to 70% alcohol. Suitably trimmed (5 mm2) samples were dehydrated with a tertiary butyl series and processed for paraffin embedding. Thick, unembedded samples were also sectioned on a sliding microtome. Transverse, radial and tangential longitudinal sections of 12–15 µm thickness were obtained with rotary and sliding microtome and stained with safranin and fast green (Johansen 1940). To obtain the length and width of vessel elements and fibres, small pieces of xylem adjacent to the cambium were macerated with Jeffrey’s solution (Berlyn & Miksche 1976) at 55 to 60 °C for 24–36 hrs, and stained with safranin to study general morphol- ogy and dimensional details. For length versus age analysis of vessel elements and fibres, small pieces of xylem were sampled at 2 mm intervals from pith to cambium and macerated. Length and diameter of the sieve tube elements were measured directly from the tangential longitudinal sections. One hundred measurements were chosen Downloaded from Brill.com09/26/2021 12:42:32PM via free access 478 IAWA Journal, Vol. 32 (4), 2011 randomly to obtain mean and standard deviation for each cell type. The numerical values given in the descriptions are expressed as minimum–maximum with standard deviations in brackets. The term ray cambium is adapted from Carlquist and Hanson (1991) for the ray cells that dedifferentiate into meristematic cells.