Cent. Eur. J. Biol. • 7(4) • 2012 • 648-654 DOI: 10.2478/s11535-012-0056-x Central European Journal of Biology Glucose inhibits the shoot bud formation in the moss Bryum billarderi Communication Arturo Martínez Zavala1,*, Netzahualcoyotl Mayek Pérez2, Analilia Arroyo Becerra1, Miguel Angel Villalobos López1 1National Polytechnic Institute, Center of Applied Biotechnology Research, 90700 Tepetitla, Mexico 2National Polytechnic Institute, Genomics Biotechnology Center, 88730 Reynosa, Mexico Received 22 November 2011; Accepted 22 April 2012 Abstract: Plant development is controlled by certain factors such as nutrient availability, environmental cues and the presence of signalling molecules. It has been proposed that phytohormones interact with sugars to modulate important processes in vascular plants. Cytokinins are key hormones because they regulate a large number of metabolic events, and sugars act as regulatory signals at several points in the life cycle. Bryum bilarderi Schwägr is a moss that was isolated by our group in the central highlands of Mexico and has demonstrated the ability to tolerate abiotic stresses. To study the effect of cytokinins and their interaction with glucose in bud induction, different concentrations of cytokinins with glucose were tested. One micromolar N-6-benzylaminopurine provided the best results for bud induction, but when 100 mM glucose was added, bud formation was inhibited. This glucose concentration also favoured the spread of the protonemal colony. These data demonstrate that N-6-benzylaminopurine is more effective than kinetin in inducing buds, and that glucose plays an important role as an inhibitory signalling molecule for the bud induction process that is mediated by cytokinins in the moss B. billarderi. Keywords: Bryophyte developmental biology • Bud formation • Bryum billarderi • Cytokinins • Glucose © Versita Sp. z o.o. Abbreviations mediation of a number of light-regulated processes such as de-etiolation [2]. Interactions between BAP - N-6-benzylaminopurine; phytohormones and sugars have been described in Glu - glucose; vascular plants. Sugars act as regulatory signals at KIN - kinetin; several points in the life cycle, and they can also MS - Murashige-Skoog medium. induce or repress genes involved in metabolic processes such as photosynthesis. They have been postulated to play an important role in different 1. Introduction developmental processes including embryogenesis, seed germination, seedling development, hypocotyl Plant growth and development is regulated by elongation, senescence induction, flowering, leaf different factors such as nutrient availability, formation, nodule growth, pollen development environmental cues and the presence of signalling and tuber and adventitious root formation [3]. molecules, including phytohormones [1]. Cytokinins Experimental data suggest that an increase in sugar are key hormones because they regulate a large levels delays seed germination and stimulates the number of processes in plants, including growth induction of flowering and senescence in at least promotion, cell division, chloroplast division, some vascular plant species. Besides these effects, senescence delay, nutrient mobilization and the soluble sugar levels can affect organ number and * E-mail: [email protected] 648 A. Martínez Zavala et al. shape [1]. In non-vascular plants, the effects from 2. Experimental Procedures the interaction between phytohormones and sugars as well as the mechanisms by which sugars influence 2.1 Plant material and growth conditions plant development are just beginning to come under Spores of B. billarderi were collected in the sub-humid study. temperate zone of Tlaxcala, Mexico (19º20’N, 98º23’W, Mosses are now becoming important model systems 2240 meters above sea level). Aseptic cultures were for the study of plant development and stress tolerance established from spores as follows: wild type B. billarderi [4-6]. Mosses are small, non-vascular plants with a sporophytes were disinfected using a 0.01% (v/v) relatively simple structure that show an alternation in sodium dichloroisocyanurate (NaDCC) solution and generations along their life cycle, which includes a then rinsed in sterile distilled water. A sterile nipper and haploid phase that generates gametes (gametophyte) a dissecting needle were used to open the capsules to and a diploid phase that produces spores via meiosis extract the spores, and the spore fluid was transferred to (sporophyte). Unlike vascular plants, the haploid the surface of full-strength Murashige and Skoog (MS) phase is dominant in mosses. Due to this feature, they medium [12], containing MS mineral salts and vitamins, are important models for genetic and developmental pH 5.75, without carbon source, solidified with 1% studies, as manipulation and genomic analysis is (w/v) agar and incubated under standard conditions (F. simpler for mosses compared with other species that Cervantes Díaz et al., unpublished results). Once that have a dominant diploid phase [7]. protonemal tissue had developed from germination of the The external application of cytokinins to mosses spores after four weeks of incubation, it was axenically has been shown to favour the transition from proliferated via asexual propagation as follows: young filamentous protonemal tissue to gametophores by protonemal tissue was dissolved in sterile distilled inducing shoot bud formation [8-10]. Further, in the water and ground with a Powergen homogenizer 125 moss Physcomitrella patens, it was reported that (Fisher Scientific, Massachusetts, USA) for 20 seconds the energy growth status promotes protonemal cell at maximum speed 3500xg. This homogenized solution differentiation; the high energy condition (addition of was inoculated on the surface of MS agar medium 150 mM glucose) favours the caulonema formation, plates and grown under standard conditions for 7 days, whereas the low energy condition induces chloronema and then these protonemal tissues were sub-cultured [11]. weekly via asexual propagation in MS medium as The aim of this study was to explore the effects described. All the experimental tissues were grown in of the interaction of glucose and cytokinins on shoot full-strength MS medium, pH 5.75, solidified with 1% bud induction and gametophore development in the (w/v) agar at 25±2ºC under long-day conditions (16 h moss Bryum billarderi Schwägr. B. billarderi Schwägr light/8 h dark) in a growth chamber. Light was provided is a moss that inhabits a sub-humid temperate zone by fluorescent tubes, and irradiance on the plant-growth in Tlaxcala, Mexico, and has the ability to tolerate shelf was 45 µmol s1 m2. different abiotic stresses during its protonemal and gametophore stages, including desiccation and 2.2 Incubation with cytokinins and glucose salinity (unpublished data). B. billarderi is a tiny non- After seven days of growth, 80 mg of protonemal tissue vascular plant which belongs to the Class Bryopsida, was dissolved in 2 mL of sterile water and ground using the Order Bryales and the Famlily Bryaceae, and it a Powergen homogenizer 125 (Fisher Scientific, MA, has been naturally found mainly in Latin America, USA) for 20 s at maximum speed 3500xg. Four ml of the and also in Australia, New Zealand and in the dissolved tissue was inoculated on permeable cellophane southern region of Africa (biodiversity occurrence discs with a 4 mm diameter, which were placed on the data published by: Australian National Herbarium, surface of MS medium, solidified with 1% (w/v) agar and Bernice Pauahi Bishop Museum, Herbarium of the incubated under standard conditions. After 21 days of University of Aarhus, Royal Botanical Garden-Madrid, incubation, six 4-mm diameter cellophane discs holding Institute of Biology - National Autonomous University the young protonemal tissue were transferred under of Mexico, National Commission for the Knowledge sterile conditions to the surface of MS agar medium and Use of Biodiversity - Mexico, Netherlands plates, without carbon source, that were supplemented Centre for Biodiversity Naturalis, Missouri Botanical with 0, 0.25, 0.50, 0.75, 1 or 1.25 µM of either Garden, South African National Biodiversity Institute, N-6-benzylaminopurine (BAP) or kinetin (KIN) (Sigma, University of Connecticut, and Institute of Natural St. Louis, MI, USA). They were grown in standard Sciences - Colombia (Accessed through GBIF Data conditions. Six days after induction, the number of Portal, data.gbif.org, 2012-04-12)). buds was observed using a microscope. To test the 649 Glucose inhibits the shoot bud formation in the moss Bryum billarderi effect of glucose on the induction of bud production, 3.2 Effect of glucose on bud formation induced six cellophane discs holding the 21-day-old protonemal by cytokinins tissue were transferred under sterile conditions to When 100 µM glucose was added to medium with the surface of MS agar medium plates containing a cytokinins, bud formation was severely inhibited at concentration of either BAP or KIN as described above, all BAP and KIN concentrations. No bud formation but now including either 0 or 100 mM of glucose. The was observed in protonemal tissue grown only in MS treated buds were grown in standard conditions. Six medium alone without cytokinins nor glucose, as well as days after induction, the number of buds was observed in MS medium with 100 mM glucose (Figures 2 and 3). using a microscope. When protonemal tissues were grown in MS solid media with 0.25 µM BAP plus glucose, the bud induction was 2.3 Quantification