HORTSCIENCE 44(7):1879–1883. 2009. plant organs. To do this, we adopted an approach that consisted, first, of carrying out anatomical characterization of these or- Localizing Starch Reserves in gans and then localizing the starch using histochemical analysis. Finally, biochemical Mandevilla sanderi (Hemsl.) Woodson analyses allowed us to quantify these re- serves. To know this quantity and the local- Using a Combined Histochemical and ization of these reserves in the plant tissue allows to assess the ability of the plant to remobilize these resources after pruning, Biochemical Approach particularly in the case of mother plant Wahiba Boutebtoub management (Latt et al., 2000), and to ensure UMR SAGAH A 462, INRA - Agrocampus Ouest, Universite´ d’Angers, 42 rue good rooting of cuttings (Eliasson, 1978). Georges Morel, B.P. 57, 49071 Beaucouze´, France Materials and Methods Michel Chevalier and Jean-Claude Mauget Plant material and culture conditions. UMR GENHORT A 1259, INRA - Agrocampus Ouest, Universite´ d’Angers, The plant material used consisted of young 42 rue Georges Morel, B.P. 57, 49071 Beaucouze´, France plants of M. sanderi, cv. Rosea fonce´, 22 1 weeks old, grown from cuttings on mother Monique Sigogne, Philippe Morel, and Gilles Galopin plants cultivated in vitro. They were planted UMR SAGAH A 462, Sciences Agronomiques Applique´es a` l’Horticulture, in 11-cm diameter plastic pots in a substrate INRA - Agrocampus Ouest, Universite´ d’Angers, 42 rue Georges Morel, B.P. consisting of a mixture of blond peat and perlite 57, 49071 Beaucouze´, France (in a 1:1 volume ratio). After a week of acclimatization in a glass greenhouse, the plants Additional index words. Apocynaceae, Mandevilla, histology, ornamental plant, starch, were placed in a growth chamber with an area tuberous roots of 10 m2 (4 · 2.50 m). A 4.5-m2 (1.5 m · 3m) subirrigation table was located in the center of Abstract. Mandevilla sanderi is a plant of tropical origin of great horticultural interest the growth room equipped with a 200-L tank for because of its abundant flowering and its persistent foliage. Vegetative propagation recycling nutrient solution (in mEqÁL–1:7.77 requires the removal of leafy branches on the mother plant to produce cuttings. This loss NO3–; 0.85 H2PO4–; 1.76 NH4+; 3.93 K+; of biomass must be compensated for by the growth of new branches thanks to the 3.30 Ca++; 1.40 Mg++; pH 5.8; electrical mobilization of reserves within the plant. Lack of knowledge about the physiology of this conductivity 1.6 mSÁcm–2). The lighting system species therefore makes it necessary to characterize its different organs both at the level consisted of 24 metallic halide lamps, Model of their anatomic organization as well as at the level of their ability to store starch. After HQIÒ-BT 400 W (OSRAMÒ, Berlin, Germany), histological characterization of the different organs (leaves, stems, and roots), starch providing average light intensity of 496 mmolÁ reserves were localized by histochemical analysis and quantified by biochemical analysis. m–2Ás–1. The photoperiod was 16 h light (from Starch grains are mainly found in the parenchymatous cortex, the parenchymatous pith 1800 HR in the evening to 1000 HR the next and xylem parenchyma cells, in tuberous roots and stems, and in the palisade and spongy morning). Room temperature was regulated by mesophyll of leaves. In 22-week-old plants, the greatest quantity of starch is found in the a climate computer with a ventilation set point leaves, whereas the tuberous roots have the highest concentration. The histological of 24 °C during the light period and 22 °C description of the different organs of Mandevilla sanderi and the localization of starch during the dark period. Plants were watered reserves allow us to assess the potential role of the different organs in plant growth and every other day with a nutrient solution. development. In the particular case of mother plant management, it is hoped that this Anatomical and histochemical analysis. knowledge will make it possible to optimize conditions for removing leafy branches. Three homogeneous plants were selected for this study. Samples were cut from roots, tuberous roots, stems, and leaves. Samples Mandevilla sanderi (Hemsl.) Woodson lotaxy, axillary racemose-type inflores- were immediately transferred after sampling (Woodson, 1933) is a plant native to Brazil, cences, and pink, infundibuliform corollas. to a fixation solution containing 4% glutaral- increasingly used in horticulture for its orna- Whereas several species of Mandevilla have dehyde in 0.2 M phosphate buffer at pH 7.2 for mental aspect, abundant and extended flow- been extensively studied for their pharmaco- 2hat4°C under vacuum. The samples were ering, persistent and glossy foliage, tolerance logical properties (Biondo et al., 2004), the rinsed in three changes of buffer, washed three to limited water availability, and resistance to physiological characteristics of this species times in distilled water, and then dehydrated many plant pests. The genus Mandevilla have not yet received adequate attention. in a graded ethanol series (70%, 80%, 95%, belongs to the family Apocynaceae, subfam- This plant is vegetatively propagated by 100%). They were infiltrated and embedded ily Apocynoideae, tribe Mesechiteae, and taking cuttings from mother plants. This in TechnovitÒ 7100 resin (2-hydroxyethyl includes more than 170 species, most of operation involves the removal of leafy methacrylate) (Heraeus Kulzer, Wehrheim, which are native to the tropical forests of branches, depriving the plant of part of its Germany) according to the procedure used South and Central America (Morales, 1998, biomass and, as a result, its glucidic com- by Kroes et al. (1998). Specimens were stored 2005; Sim¨oes et al., 2007). Mandevilla sand- pounds. For Platanus acerifolia, this loss of at 37 °C. Three-micron thick sections were eri is characterized by its woody, volubile biomass has an impact on the plant’s ability cut with a Reichert-Jung 1140 microtome stems, persistent foliage with decussate phyl- to continue its growth (Haddad et al., 1995). (Reichert-Jung, Nussloch, Germany) and Its subsequent development thus requires the mounted on clean glass slides. mobilization of reserves present in its differ- The following two staining methods were Received for publication 5 May 2009. Accepted ent organs. The lack of knowledge about the used: 1) periodic acid/Schiff: sections were for publication 31 Aug. 2009. physiology of this species therefore makes it treated in periodic acid for 20 min, stained with We thank Lannes et Fils, SA, Marie Tellier, and Catherine Bernard (UMR-GenHort) for their con- necessary to characterize at the level of their Schiff’s reagent for 30 min, differentiated with tribution to this study and Gail Wagman for the anatomic organization as well as at the level sulphuric water at 1%, rinsed with distilled English translation of the text. of their ability to store starch. water, and mounted in a synthetic resin. 1To whom reprint requests should be addressed; The purpose of this study was to localize Schiff’s reagent stained carbohydrate com- e-mail [email protected]. and to quantify starch reserves in the various pounds red. It made it possible to visualize cell HORTSCIENCE VOL. 44(7) DECEMBER 2009 1879 Figs. 1–5. Morphologic structure of Mandevilla sanderi. Fig. 1. Aerial apparatus. Fig. 2. Volubile stem. Fig. 3. Young leaves. Fig. 4. Mature leaf. Fig. 5. Adventitious root system. (A) Tuberous roots; (B) fine roots. Figs. 6–7. Anatomical structure of young roots of Mandevilla sanderi. Figs. 6– 7. Transverse sections stained with Schiff’s reagent Fig. 6. Transverse section of a young root. Fig. 7. Cortical tissue. (A) Exodermis; (B) parenchyma cells; (C) phloem; (D) xylem; (E) pericycle; (F) endodermis; (G) exodermis; (H) laticifer; arrow (<): starch; Bar (–––): 100 mm. walls and starch grains; 2) iodine potassium iodide: sections were placed in the solution for 2 h and then rinsed with distilled water and mounted in synthetic resin. Starch grains were specifically stained purplish blue. Samples were observed using a BH-RFC OlympusÒ microscope (Olympus, Tokyo, Japan) combined with a Sony 3CCD camera (Sony, Tokyo, Japan). Biochemical analysis. Nine plants were separated into different compartments: roots, tuberous roots, stems, leaves, and inflores- cences. These compartments were quickly immersed in liquid nitrogen and then freeze- dried for 48 h (Leybold Heraeus-GT-2; Ley- bold Didactic GmbH, Hu¨rth, Germany). The dry weight obtained after freeze-drying cor- responded to the weight of the apparent dry matter (ADM) (Gomez et al., 2003). A fine powder was obtained by placing the samples in a ball grinder (RetschÒ MM301; Retsch, Haan, Germany), maintaining a low temper- ature, and then bringing them back to room temperature in a dessicator before samples were weighed for assaying. Aliquots of sam- ples of 300 mg of dry powder were prepared. After extraction of soluble sugars with 80% ethanol at 70 °C, the residue was recovered and dried in a ventilated drying oven at 80 °C. Figs. 8–12. Anatomical structure of tuberous root of Mandevilla sanderi. Figs. 8–10. Transverse sections Dispersion and hydrolysis of starch using stained with Schiff’s reagent. Fig. 8. transverse section of a tuberous root. Fig. 9. Cortical tissue. Fig. a-amylase enzymes at 100 °C and amyloglu- 10. Vascular cylinder. Figs. 11–12. Transverse sections stained with iodine potassium iodide. Fig. 11. cosidase at 50 °C (Megazyme International Starch localization. Fig. 12. Cortical tissue. (A)Corklayer;(B) parenchyma cells; (C) secondary phloem; Ireland Ltd., Bray, UK) freed the glucose, (D) secondary xylem; (E)laticifers;(F) ray parenchyma cells; arrow (<): starch; Bar (–––): 100 mm. 1880 HORTSCIENCE VOL. 44(7) DECEMBER 2009 which was then quantified using the colori- considerably increased. Xylem vessels are chlorophyllous spongy mesophyll cells (Fig. metric method with anthrone in sulphuric laid out in radial rays separated by the 19).