Biotechnology Summit 2012 Biotechnology Summit

International Fundation for Biotechnology Research & Early Stimulation in the Culture of Health, Nutrition, Sport, Art Science, Technology & Society A.C. Nonproﬁt Organization

Biotechnology Summit 2012 Mérida, Yucatán

Mexico

Editors Fabián Fernández-Luqueño Fernando López-Valdez Susana Lozano-Muñiz

ISBN WEB VERSION: 978-607-9023-12-6

Appropriate form to cite the articles included in this compendious (example): Villanueva-Lizama LE. Dumonteil E 2012. Immunogenic salivary proteins of Triatomadimidiata, a vector of Chagas disease. In: Fernández-Luqueño F, López-Valdez F, Lozano-Muñiz S. (Eds.). Biotechnology Summit 2012, Yucatán México. pp. 1-5. 12-21 March 2012.

This compendious of Biotechnology Summit 2012 Are property and responsibility of Authors. All or any part of this publication may be reproduced or transmitted, by any means, electronic or mechanical (Including photocopying, recording or any recovery system and storage), and must be included with the corresponding citation of this compendious and their authors. International Foundation for Biotechnology Research & Early Stimulation in the Culture of Health, Nutrition, Sport, Art, Science, Technology & Society A.C. Nonprofit Organization (IFBRESCHSASTSAC) International Foundation for Biotechnology Research & Early Stimulation in the Culture of Health, Nutrition, Sport, Art, Science, Technology & Society A.C. Nonprofit Organization D.R. © 1st Edition Centro de Investigación y de Estudios Avanzados del I.P.N. Cinvestav 2012 Publisher Cinvestav, AV. I.P.N. 2508 07360, MÉXICO, D.F.

WEB version ISBN: 978-607-9023-12-6

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ANALYSIS OF THE INSECTICIDAL ACTIVITIES AND PERSISTENCE OF ESSENTIAL OIL … OF AN AROMATIC PLANT (CITRUS AURANTIFOLIA) Oko, Augustine Okpani, Ekigbo, Justus Chinagorum, Ubi, Benjamin Ewa, Idike, Francis.

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PHENOLOXIDASE ACTIVITY BY PERIPLANETA AMERICANA (BLATTARIA: … BLATTIDAE) EXPOSED TO BEAUVERIA BASSIANA Patricia Tamez-Guerra, Gabriela Damas-Buenrostro, Juan Manuel BallesterosTorres, José Alberto Valadéz-Lira, Ricardo Gomez-Flores, Cristina RodríguezPadilla.

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PATHOGENICITY OF ISARIA FUMOSOROSEA ON IMMATURE WHITEFLY BEMISIA … TABACI (HEMIPTERA: ALEYRODIDAE) Esaú Ruiz Sánchez, Wilberth Chan Cupul, Arturo Reyes Ramírez, Jairo Cristóbal Alejo, Horacio S. Ballina Gómez, Alfonzo Pérez Gutiérrez, Joel Lara Reyna.

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GERMINATION AND PLANT DEVELOPMENT OF PROTOCORM-LIKE BODIES OF … ENCYCLIA YUCATANENSE AFTER MATURATION AND DESICCATION TREATMENTS Briceño-Dominguez Diego Ramón, Cruz-Santander Ivonne, Nahuat-Dzib Sara Luz, José Luis Giorgana Figueroa.

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DNA LIBRARY …

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BACTERIAL DIVERSITY AND CONSTRUCTION OF A METAGENOMIC FROM WHEAT RHIZOSPHERE

Gustavo Santoyo, Rocío Hernández-León, Ma. del Carmen Orozco-Mosqueda and Eduardo Valencia-Cantero … BIOENERGY AS AN EXCELLENT CHOICE OF RENEWABLE ENERGY Angelina González-Rosas a, Juan Marcelo Miranda-Gómez a, Germán ReséndizLópez a, Luís García-Lechuga a, Fabián Fernández-Luqueño

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…

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BIOTECHNOLOGY-DERIVED PRODUCTS FOR INSECT PEST CONTROL Patricia Tamez-Guerra, Robert W. Behle

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STUDY OF THE FERMENTATIVE CAPACITY AND ETHANOL PRODUCTION OF TWO MICROORGANISMS ISOLATED FROM BOVINE RUMEN

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IMMUNITY RESPONSE BY SECOND- AND FOURTH-INSTAR LARVAE OF FOUR LEPIDOPTERAN PESTS AFTER BACILLUS THURINGIENSIS EXPOSURE José Alberto Valadéz-Lira, Gabriela Damas-Buenrostro, Juan Manuel AlcocerGonzález, Gricelda Núñez-Mejía, Brenda Oppert, Ricardo Gomez-Flores, Cristina Rodríguez-Padilla, Patricia Tamez-Guerra

Raziel Estrada-Martínez, Tania González-Flores, María de los Ángeles SánchezContreras, Ingrid Rodríguez-Buenfil

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BIOTECHNOLOGY SUMMIT 2012 YUCATÁN, MEXICO

Briceño-Dominguez et al.

GERMINATION AND PLANT DEVELOPMENT OF PROTOCORM-LIKE BODIES OF ENCYCLIA YUCATANENSE AFTER MATURATION AND DESICCATION TREATMENTS

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Briceño-Dominguez Diego Ramón1, Cruz-Santander Ivonne1, Nahuat-Dzib Sara Luz2 and José Luis Giorgana Figueroa2

Laboratorio de Ciencias Básicas. Instituto Tecnológico Superior de Felipe Carrillo Puerto. Carr. Vigía 2 Chico- F.C.P Km. 1.5 Col. Centro. C.P. 77200. Felipe Carrillo Puerto, Quintana roo. Laboratorio de Biotecnología vegetal. Instituto Tecnológico de Mérida. Av. Tecnológico km. 4.5 S/N C.P. 97118 Mérida, Yucatán. Corresponding author email: [email protected]

Abstract: Orchid population in Yucatan peninsula has been considerably reduced environmental and anthropogenic causes. One tool for recover the orchid habitat and reduce this pressure is using plant biotechnologies as plant tissue culture and artificial seed technology. This research was conducted to develop a methodology to produce artificial seed of the orchid Encyclia yucatanense assesing the influence of the composition of maturation medium based in Murashige and Skoog (MS) medium added with abscisic acid (ABA), proline (PRO) and polyethylene glycol (PEG). MS medium added with plant growth regulators was used as control. We studied germination and plant development of protocorm-like bodies (PLBs) after maturation and desiccation. We observed that maturation medium improved in PLBs tolerance to desiccation. Germination of PLBs was not significantly different from the control however; plant development was significantly lower than control. After desiccation, PLBs showed humidity values between 48-57%, while humidity content of PLBs from control treatment was close to 87%, germination and plant development of PLBs of control treatment was significantly lower than maturation medium treatments. Best results were obtained with maturation medium added with 7.5 µM, 15 µM and 5% of ABA, PRO and PEG, respectively. Keywords: Orchid ● Artificial seed ● Maturation Introduction: Orchids show a wide range of diversity of flowers (color, size, shape, etc.). They exhibit a high price in both national and international markets. In Mexico, natural orchids habitat has been damaged by natural disasters, cities development, and also caused by people that take adult plants for sell them. In vivo vegetative propagation are time consuming and expensive because propagation of orchids from seeds is undesirable due to the heterozygosity of seed, the seed size, presence of reduced endosperm, also the requirement of an association with mycorrhizal fungi. However, Orchids can be multiplied by micropropagation. Artificial seed could be defined as an analogous of zygotic seed; it can be presented as desiccated somatic embryo without cover; desiccated somatic embryo coated with a water soluble resin; somatic embryo coated with alginate gel and somatic embryo into a gel called as fluid drilling (Fujii et al, 1987). Most limiting aspect of artificial seed technology is the production of vigorous somatic embryos also, for the market competition the artificial seed need to have a good and quick germination and develop in plant as a natural seed (Anandarajah & McKersie, 1990). The objective of this research was to asses conditions for preservation of the Yucatan’s peninsula orchid’s germoplasm as artificial seed, for the reestablishment of damaged ecosystems or for a commercial aim. We assessed the effect of addition abscisic acid (ABA), proline (PRO) and polyethylene glycol (PEG). in a basal MS medium defined as maturation medium on germination and plant development after maturation and desiccation of PLBs.

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Material and methods: Production and maintenance of Protocorm-Like Bodies (PLBs) PLBs from E. yucatanense were obtained from previous works and these were reproduced using Murashige and Skoog (1962) media added with myo-inositol (100mg L-1), thiamine (4mg L-1), cysteine (50mg L-1), sucrose (30g L-1) and plant growth regulators: naphtaleneacetic acid, indoleacetic acid and bencilaminopurine used at 2 mg L-1. Incubation for maintenance and experiments of PLBs were using a plant growth room at 25-27 ºC, relative humidity of 80%, fluorescents lamps as light source (45-60 µmol m-2s-1) and photoperiod light:dark (16:8h) Experimental design For maturation treatments, MS media was added with ABA, PRO and PEG in different levels (Table 1). Samples of 300 of PLBs were used in each treatment in triplicate. Table 1. Levels of ABA, PRO and PEG used in the maturation medium. Treatment ABA(µM) PRO(µM) PEG (%) Control 0 0 0 T-1 7.5 10 5 T-3 7.5 15 5 T-5 15 10 5 T-7 15 15 5 Control treatment was same medium used for the production of PLBs. Maturation and Desiccation of PLBs Maturation consisted in grow PLBs during 15 days in each treatment and after this time, PLBs were subjected to gradual desiccation by applying 6 stages using individually the hygroscopic salts K2SO4; Na2CO3; NaCl; NH4NO3; Ca(NO3)2 and CaCl2. All experimental treatments remained 24h in each desiccation stage and humidity of PLBs was calculated. After maturation and desiccation, germination and plant development was assessed. Also, dry weight was determinate after desiccation treatment. Results and discussion: After PLBs maturation treatments, detention of growth was visually observed in 87.7±5.3 % while control treatment did not affect differentiation and conversion to plant. Compared with zygotic embryogenesis, where the embryo into the seed is in quiescence and dormancy status and this grow up only if the external conditions are optimal, somatic embryos do not stop its growth (Senaratna et al, 1990) The presence of PEG in maturation medium allowed the PLBs diminish its humidity content with a significant difference compared with control, humidity content of treatments was approximate 67-57%, after gradual desiccation of PLBs was close 57-48% (Figure 1). Germination after maturation was similar in all treatments and values were between 6570% and it was not significant differences between treatments and control (MS). After desiccation, germination was significantly affected in control and T-7 treatment, this may be caused by salt stress that could had an effect in viability of PLBs, because T-7 medium had highest ABA and PRO contents (15µM and 15µM, respectively) (figure 2). PLBs reduced its growth and development process close to 15-40%, considerably lower than control, this effect could be caused by ABA presence in the maturation medium, it is know that ABA regulates key events during seed formation, such as the deposition of storage reserves, prevention of precocious germination, acquisition of desiccation tolerance, and

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induction of primary dormancy (Kermode, 2005). After desiccation process, percentage of plant growth and development increased considerably compared to control (figure 3).

Figure 1. Humidity content after each desiccation stages of PLBs treated with 4 different maturation media and MS media used as control.

Figure 2. Germination (%) of PLBs treated with maturation media after 15 days maturation period (D-0) and after gradual desiccation (D-6). Different letter indicate significant differences using two-way ANOVA (α=0.05).

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Figure 3. Plant development (%) from PLBs treated with maturation media after 15 days maturation period (D-0) and after gradual desiccation (D-6). Different letter indicate significant differences using two-way ANOVA (α=0.05). Plant development after desiccation process was increased with the treatments. Also, dry weight of PLBs from treatments was significantly higher than control treatment; this effect was due to the presence of proline in maturation media. It is well known that proline have positive effects on enzyme and membrane integrity along with adaptive roles in mediating osmotic adjustment in plants grown under stress conditions (Ashraf & Foolad, 2007). Proline is accumulated during osmotic stress causing increment of dry weight (Hoekstra et al, 2001), as we observed in PLBs treated (figure 4).

Figure 4. Dry weight of PLBs treated with maturation media added with ABA, PRO and PEG. Different letter indicate significant differences using one-way ANOVA (α=0.05). Conclusions: Our finding shows the importance of define a maturation treatment to achieve a method for production of Yucatan’s orchids artificial seed. The PLBs maturation was achieved with Murashigue and Skoog culture media added with ABA (10 or 15µM), PRO (7.5µM) and PEG (5 %) followed of gradual desiccation stages using hygroscopic salts. References:

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Anandarajah K, McKersie BD 1990. Enhanced vigour of dry somatic embryos of Medicago sativa L. with increased sucrose. Plant Science 71:261-266. Ashraf M, Foolad MR 2007. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany 59(2):206–216. Fujii JA, Slade DT, Redenbaugh K, Walker KA 1987. Artificial seeds for plant propagation. Trends in Biotechnology 5(12):335-339. Hoekstra FA, Golovina EA, Buitink J 2001. Mechanisms of plant desiccation tolerance. Trends Plant Science 6:431-438. Kermode AR 2005. Role of Abscisic Acid in Seed Dormancy. Journal of Plant Growth Regulation 24(4):319–344. Murashige T, Skoog F 1962. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum 15(3):473–497. Senaratna T, McKersie BD, Bowley SR 1990. Artificial seeds of alfalfa (Medicago sativa L.). Induction of desiccation tolerance in somatic embryos. In Vitro Cellular & Developmental Biology-Plant 26(1): 85–90.

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