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Manual for the in Vitro Culture of Agaves. Common Fund For OCCASION This publication has been made available to the public on the occasion of the 50th anniversary of the United Nations Industrial Development Organisation. DISCLAIMER This document has been produced without formal United Nations editing. The designations employed and the presentation of the material in this document do not imply the expression of any opinion whatsoever on the part of the Secretariat of the United Nations Industrial Development Organization (UNIDO) concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries, or its economic system or degree of development. Designations such as “developed”, “industrialized” and “developing” are intended for statistical convenience and do not necessarily express a judgment about the stage reached by a particular country or area in the development process. Mention of firm names or commercial products does not constitute an endorsement by UNIDO. FAIR USE POLICY Any part of this publication may be quoted and referenced for educational and research purposes without additional permission from UNIDO. However, those who make use of quoting and referencing this publication are requested to follow the Fair Use Policy of giving due credit to UNIDO. CONTACT Please contact [email protected] for further information concerning UNIDO publications. For more information about UNIDO, please visit us at www.unido.org UNITED NATIONS INDUSTRIAL DEVELOPMENT ORGANIZATION Vienna International Centre, P.O. Box 300, 1400 Vienna, Austria Tel: (+43-1) 26026-0 · www.unido.org · [email protected] UNITED NATIONS COMMON FUND INDUSTRIAL FOR COMMODITIES DEVELOPMENT ORGANIZATION - --' Com man Fund for Com mod ities Technical Paper no. 38 Manual for the in Vitro Culture of Agaves UNITED NATIONS INDUSTRIAL DEVELOPMENT ORGANIZATION Vienna 2004 Copyright © 2004 by the Common Fund for Commodities (CFC) First published October 2004 The contents of this document may not be reproduced, stored in data retrieval systems or transmitted in any form or means without prior written permission of the Common Fund for Commodities. Reasonable extracts may be made for the purpose of non-commereial use provided that the Common Fund for Commodities is acknowledged. This Manual has been produced by a team of experts in the framework of a Common Fund project (CFCIFIGHFI07 - Product and Market Development of Sisal and Henequen Products) financed at the request of the Food and Agriculture Organization of the United Nations (FAO) Intergovernmental Group on Hard Fibres (FIGHF). The project was implemented by the United Nations Industrial Development Organization (UNIDO) under the supervision of the FIGHF. Designations employed and the presentation of material in this publication do not imply the expression of any opinion whatsoever on the part of the Common Fund for Commodities or of the Secretariat of the United Nations Industrial Development Organization or of the Food and Agriculture Organization of the United Nations concerning the legal status of any counliy, territory, city or area, or of its authorities, or concerning the delimitation of its frontiers or boundaries. The opinions, figures and estimates set forth are the responsibility of the authors -- - and-iihoulo not necessarilylJe consiuerea aEeflect\ng 'the views 0t carrying the endorsement of CFC, UNlDO or FAO. The mention of firm names or commercial products 'does not imply endorsement by CFC, UNIDO or FAO. This document has not been formally edited: ," The Manual for the in Vitro Cultur,e of Agaves is the second document resulting from the project which is included in the CFC Technical Paper series. The first publication was entitled: SISAL - Past Research Results and Present Production Practices in East Africa (CFC Technical Paper no. 8). Common Fund for Commodities telephone: (++3120) 575 4949 Stadhouderskade 55 fax: . (++31 20) 676 0231 1072 AB Amsterdam e-mail: [email protected] The Netherlands web site: www.common-fund.org United Nations Industrial Development Organization Vienna International Centre telephone: (++43 I) 26026 3945 P.O. Box 300 fax: (++431) 21346 3892 A-1400 Vienna e-mail: [email protected] Austria web site: www.unido.org Food and Agriculture Organization of the United Nations Commodities and Trade Division telephone: (++39) 06 5705 420 I Viale delle Terme di Caracalla fax: (++39) 06 5705 4495 00 I00 Rome e-mail: [email protected] Italy http://www.fao.org/es/ESC/ Manual for the in VitroCulture of Agaves Compiled by 1 2 Robert, M.L. ,2, Herrera-Herrera, J.L.2, Herrera-Alamillo, M.A , Quijano-Ramayo, A.2 and Balam Uc E.2 Other Contributors: 3 z Dr. Amarella Eastmond ; lng. Gaston Herrera Herrera ; Dr. Felipe Barahona; Z z Q.B.A. Ileana Borges Argaez , BioI. Felipe Barredo . The contribution of the following persons who indirectly helped through useful scientific discussions on the technical problems of the techniques is acknowledged: 4 4 Mr. Shabani Hamisi , Mrs. Beatrice E. Mlal, Mrs. Lady A. Swai , Mr. Joseph 5 6 6 Tipape ; Mr.Phillip Njoroge ; Ms. Elizabeth Macharia ; Ms. Salome Nthenya 6 6 Kivuva ; Mr. Stephen Fwamba Khisa and Mr. John Kamau (l) International consultant and Research scientist (2) Unidad de Biotecnologia, Centro de Investigacion Cientifica de Yucatan A.C. Mexico. (3) Unidad de Ciencias Sociales,Centro de Investigaciones Regionales Dr Hydeyo Noguchi, Universidad Autonoma de Yucatan, Mexico. (4) MTC Laboratory, ARI Mlingano, Tanga, Tanzania. (5) Deputy Manager, Kenya Sisal Board, Nairobi, Kenya. (6) Plant Tissue Culture Laboratory KEPHIS, Muguga, Kenya. III Foreword Like many other natural fibres, sisal is facing strong competition from synthetic materials. In order to retain its current market share and to re-gain its lost markets, the sisal industry needs to increase both its production efficiency as well as to develop new applications and open up new markets. --The-Gammon ·Fund-for-Commodities is providing-support to the-sisal-sector,-- through its financing of several projects, to attain the same. The Fund-supported project "Product and Market Development of Sisal and Henequen Products" (CFCjFIGHFjOl) has been implemented by the United Nations Industrial Development Organization in Kenya and Tanzania during the years 1997 - 2004. The designated international commodity body for hard fibres, the FAO Intergovernmental Group on Hard Fibres (FIGHF), supervised its implementation. One of the components of this project focused on the improvement of the vegetative material which is the basis of the quality of the sisal plants. The emphasis was both on developing a reliable method of producing high quality, disease-free-plantlets--as-well-as by-passing-thecostly-and time consuming stage of propagation through bulbils in designated nurseries. The method of Meristematic Tissue Culture (MTC) was used in this project in sisal laboratories in both Kenya and Tanzania, with the objective of multiplying promising sisal cultivars to promote the production of standard planting materials and to shorten the time needed by breeders to produce new varieties for commercial production. In order to document the MTC practices developed and used in the project, the current Manualwas prepared by the project. It is hoped that the Manual will provide insights in the techniques and methods applied and serve as a reference document for researchers in the field of sisal (re-) production. Amb. Ali Mchumo Managing Director Common Fund for Commodities IV Contents Foreword iv Acronyms I Abbreviations ix Preface xi Executive Summary 2 Chapter 1 Introduction 6 1.1 The uses of agaves 6 1.2 The need for research and innovation 7 1.3 The role of plant biotechnology 8 Chapter 2 Genetic Improvement 10 2.1 Introduction 10 2.2 The reproductive biology of agaves 11 2.2.1 Sexual reproduction 2.2.2 Asexual propagation 2.3 Problemsassociatedwith the genetic improvement of agaves 16 2.4 Genetic improvement of agaves 16 2.4.1 Is there enough genetic variability in the populationsof agaves? 17 2.4.2 Selection 17 2.4.3 Production of hybrids through controlled polinization 18 2.4.4 Open pollination 19 2.5 Biotechnologicalalternatives 20 Chapter3 Agave Tissue Culture Research 22 3.1 A brief introduction to Micropropagation 22 3.2 The organogenic and embryogenic pathways 23 3.2.1 Callusformation 23 3.2.2 Direct organogenesis 23 3.2.3 Indirect organogenesis 24 3.2.4 Somatic embryogenesis 24 3.3 Agave tissue culture 24 v 3.3.1 Micropropagation through direct organogenesis 24 3.3.2 Micropropagation through indirect organogenesis 25 3.3.3 Micropropagation through direct somatic embryogenesis 26 3.3.4 Micropropagation through indirect somatic embryogenesis 26 3.4 Meristem culture 27 3.5 Present trends in Agave biotechnology research 28 "- ------- --- ---" --- Chapter 4 Guidelines for the Establishment of a Micropropagation Protocol for Agaves "32 4.1 Objectives 32 4.2 Establishing a protocol 32 4.3 A method for an efficient micropropagation of Agave species 33 4.3.1 Selection of the "elite" mother plants 34 4.3.2 Sources of explants 35 4.3.2.1 Rhizomes 35 4.3.2.2 Other sources of explants 37 4.3.3 Pre-conditioning of donor plants ""37 4.3.4 Extraction--6nhe meristematiC"tissues 38 4.3.5 Disinfestation of the explants 40 4.3.6 Cutting of explants 41 4.3.7 Induction: Culturing the explants 42 4.3.7.1 Separation of the induced plantlets 45 4.3.8 Multiplication 45 4.3.9 Growth 46 4.3.10 In vitropre-adaptation 48 4.3.11 In vitrorooting 49 4.4 Common Problems 50 4.4.1
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