Plant tissue culture engineering

Tai Lieu Chat Luong PLANT TISSUE CULTURE ENGINEERING FOCUS ON BIOTECHNOLOGY Volume Series Editors MARCEL HOFMAN Centre for Veterinary and Agrochemical Research, Tervuren, Belgium JOZEF ANNÉ Rega Institute, University of Leuven, Belgium Volume Editors S DUTTA GUPTA Department of Agricultural and Food Engineering, Indian Institute of Technology, Kharagpur, India YASUOMI IBARAKI Department of Biological Science, Yamaguchi University, Yamaguchi, Japan COLOPHON Focus on Biotechnology is an open-ended series of reference volumes produced by Springer in co-operation with the Branche Belge de la Société de Chimie Industrielle a.s.b.l The initiative has been taken in conjunction with the Ninth European Congress on Biotechnology ECB9 has been supported by the Commission of the European Communities, the General Directorate for Technology, Research and Energy of the Wallonia Region, Belgium and J Chabert, Minister for Economy of the Brussels Capital Region Plant Tissue Culture Engineering Edited by S DUTTA GUPTA Department of Agricultural and Food Engineering, Indian Institute of Technology, Kharagpur, India and YASUOMI IBARAKI Department of Biological Science, Yamaguchi University, Yamaguchi, Japan A C.I.P Catalogue record for this book is available from the Library of Congress ISBN-10 ISBN-13 ISBN-10 ISBN-13 1-4020-3594-2 (HB) 978-1-4020-3594-4 (HB) 1-4020-3694-9 (e-book) 978-1-4020-3694-1 (e-book) Published by Springer, P.O Box 17, 3300 AA Dordrecht, The Netherlands www.springer.com Printed on acid-free paper All Rights Reserved © 2006 Springer No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work Printed in the Netherlands FOREWORD It is my privilege to contribute the foreword for this unique volume entitled: “Plant Tissue Culture Engineering,” edited by S Dutta Gupta and Y Ibaraki While there have been a number of volumes published regarding the basic methods and applications of plant tissue and cell culture technologies, and even considerable attention provided to bioreactor design, relatively little attention has been afforded to the engineering principles that have emerged as critical contributions to the commercial applications of plant biotechnologies This volume, “Plant Tissue Culture Engineering,” signals a turning point: the recognition that this specialized field of plant science must be integrated with engineering principles in order to develop efficient, cost effective, and large scale applications of these technologies I am most impressed with the organization of this volume, and the extensive list of chapters contributed by expert authors from around the world who are leading the emergence of this interdisciplinary enterprise The editors are to be commended for their skilful crafting of this important volume The first two parts provide the basic information that is relevant to the field as a whole, the following two parts elaborate on these principles, and the last part elaborates on specific technologies or applications Part deals with machine vision, which comprises the fundamental engineering tools needed for automation and feedback controls This section includes four chapters focusing on different applications of computerized image analysis used to monitor photosynthetic capacity of micropropagated plants, reporter gene expression, quality of micropropagated or regenerated plants and their sorting into classes, and quality of cell culture proliferation Some readers might be surprised by the use of this topic area to lead off the volume, because many plant scientists may think of the image analysis tools as merely incidental components for the operation of the bioreactors The editors properly focus this introductory section on the software that makes the real differences in hardware performance and which permits automation and efficiency As expected the larger section of the volume, Part covers Bioreactor Technologythe hardware that supports the technology This section includes eight chapters addressing various applications of bioreactors for micropropagation, bioproduction of proteins, and hairy root culture for production of medicinal compounds Various engineering designs are discussed, along with their benefits for different applications, including airlift, thin-film, nutrient mist, temporary immersion, and wave bioreactors These chapters include discussion of key bioprocess control points and how they are handled in various bioreactor designs, including issues of aeration, oxygen transport, nutrient transfer, shear stress, mass/energy balances, medium flow, light, etc Part covers more specific issues related to Mechanized Micropropagation The two chapters in this section address the economic considerations of automated micropropagation systems as related to different types of tissue proliferation, and the use of robotics to facilitate separation of propagules and reduce labour costs Part 4, Engineering Cultural Environment, has six chapters elaborating on engineering issues related to closed systems, aeration, culture medium gel hardness, dissolved oxygen, v Foreword photoautotrophic micropropagation and temperature distribution inside the culture vessel The last part (Part 5) includes four chapters that discuss specific applications in Electrophysiology, Ultrasonics, and Cryogenics Benefits have been found in the use of both electrostimulation and ultrasonics for manipulation of plant regeneration Electrostimulation may be a useful tool for directing signal transduction within and between cells in culture Ultrasound has also applications in monitoring tissue quality, such as state of hyperhydricity Finally the application of engineering principles has improved techniques and hardware used for long-term cryopreservation of plant stock materials Readers of this volume will find a unique collection of chapters that will focus our attention on the interface of plant biotechnologies and engineering technologies I look forward to the stimulation this volume will bring to our colleagues and to this emerging field of research and development! Gregory C Phillips, Ph D Dean, College of Agriculture Arkansas State University vi PREFACE Plant tissue culture has now emerged as one of the major components of plant biotechnology This field of experimental botany begins its journey with the concept of ‘cellular totipotency’ for demonstration of plant morphogenesis Decades of research in plant tissue culture has passed through many challenges, created new dreams and resulted in landmark achievements Considerable progress has been made with regard to the improvement of media formulations and techniques of cell, tissue, organ, and protoplast culture Such advancement in cultural methodology led many recalcitrant plants amenable to in vitro regeneration and to the development of haploids, somatic hybrids and pathogen free plants Tissue culture methods have also been employed to study the basic aspects of plant growth, metabolism, differentiation and morphogenesis and provide ideal opportunity to manipulate these processes Recent development of in vitro techniques has demonstrated its application in rapid clonal propagation, regeneration and multiplication of genetically manipulated superior clones, production of secondary metabolites and ex-situ conservation of valuable germplasms This has been possible not only due to the refinements of cultural practices and applications of cutting-edge areas of molecular biology but also due to the judicious inclusion of engineering principles and methods to the system In the present scenario, inclusion of engineering principles and methods has transformed the fundamental in vitro techniques into commercially viable technologies Apart from the commercialization of plant tissue culture, engineering aspects have also made it possible to improve the regeneration of plants and techniques of cryopreservation Strategies evolved utilize the disciplines of chemical, mechanical, electrical, cryogenics, and computer science and engineering In the years to come, the application of plant tissue culture for various biotechnological purposes will increasingly depend on the adoption of engineering principles and better understanding of their interacting factors with biological system The present volume provides a cohesive presentation of the engineering principles and methods which have formed the keystones in practical applications of plant tissue culture, describes how application of engineering methods have led to major advances in commercial tissue culture as well as in understanding fundamentals of morphogenesis and cryopreservation, and focuses directions of future research, as we envisage them We hope the volume will bridge the gap between conventional plant tissue culturists and engineers of various disciplines A diverse team of researchers, technologists and engineers describe in lucid manner how various engineering disciplines contribute to the improvement of plant tissue culture techniques and transform it to a technology The volume includes twenty four chapters presenting the current status, state of the art, strength and weaknesses of the strategy applicable to the in vitro system covering the aspects of machine vision, bioreactor technology, mechanized micropropagation, engineering cultural environment and physical aspects of plant tissue engineering The contributory chapters are written by international experts who are pioneers, and have made significant contributions to vii Preface this emerging interdisciplinary enterprise We are indebted to the chapter contributors for their kind support and co-operation Our deepest appreciation goes to Professor G.C Phillips for sparing his valuable time for writing the Foreword We are grateful to Professor Marcel Hofman, the series editor, ‘Focus on Biotechnology’ for his critical review and suggestions during the preparation of this volume Our thanks are also due to Dr Rina Dutta Gupta for her efforts in checking the drafts and suggesting invaluable clarifications We are also thankful to Mr V.S.S Prasad for his help during the preparation of camera ready version Finally, many thanks to Springer for their keen interest in bringing out this volume in time with quality work S Dutta Gupta Y Ibaraki Kharagpur/Yamaguchi, January 2005 viii TABLE OF CONTENTS FOREWORD……………………………………………………………………… ….v PREFACE………………………………………………………………………… …vii TABLE OF CONTENTS………………………………………………………………1 PART 13 MACHINE VISION 13 Evaluation of photosynthetic capacity in micropropagated plants by image analysis 15 Yasuomi Ibaraki 15 Introduction 15 Basics of chlorophyll fluorescence 16 Imaging of chlorophyll fluorescence for micropropagated plants 18 3.1 Chlorophyll fluorescence in in vitro cultured plants 18 3.2 Imaging of chlorophyll fluorescence 21 3.3 Imaging of chlorophyll fluorescence in micropropagated plants 22 Techniques for image-analysis-based evaluation of photosynthetic capacity 25 Estimation of light distribution inside culture vessels 26 5.1 Understanding light distribution in culture vessels 26 5.2 Estimation of light distribution within culture vessels 26 Concluding remarks 27 References 28 Monitoring gene expression in plant tissues 31 John J Finer, Summer L Beck, Marco T Buenrostro-Nava, Yu-Tseh Chi and Peter P Ling 31 Introduction 31 DNA delivery 32 2.1 Particle bombardment 32 2.2 Agrobacterium 33 Transient and stable transgene expression 33 Green fluorescent protein 34 4.1 GFP as a reporter gene 34 4.2 GFP image analysis 35 4.3 Quantification of the green fluorescence protein in vivo 36 Development of a robotic GFP image acquisition system 37 5.1 Overview 37 5.2 Robotics platform 37 5.3 Hood modifications 39 5.4 Microscope and camera 40 5.5 Light source and microscope optics 40 Automated image analysis 41 6.1 Image registration 41 6.2 Quantification of GFP 43 Acoustic characteristics of plant leaves using ultrasonic transmission waves [2] Fukuhara, M and Sampei, A (1993) Elastic moduli and internal frictions of carbon and stainless steels as a function of temperature Iron and Steel Inst J Int 33: 508-512 [3] Fukuhara, M and Sampei, A (1994) Low-temperature elastic moduli and dilational and shear internal frictions of superconducting ceramic GdBa2Cu3O7-į Phys Rev B49: 13099-13105 [4] Numata, H and Fukuhara, M (1997) Low-temperature elastic anomalies and heat generation of deuterated palladium Fus Tech 31: 300-310 [5] Fukuhara, M.; Degawa, T.; Okushima, L and Homma, T (2000) Propagation characteristics of leaves using ultrasonic transmission waves Acoust Lett 24:70-74 [6] Fukuhara, M (2002) Acoustic characteristics of botanical leaves using ultrasonic transmission waves Plant Sci 162: 521-528 [7] Kinra, V K and Dayal, V (1988) A new technique for ultrasonic non-destructive evaluation of thin specimen J Exp Mech 28: 288-297 [8] Kinra, V K and Zhu, C (1993) Time-domain ultrasonic NDE of the wave velocity of a subhalfwavelength elastic layer J Test Eval 21: 29-35 [9] Wan, M.; Jiang, B and Cao, W (1997) Direct measurement of ultrasonic velocity of thin elastic layers J Acoust Soc Am 101:626-628 [10] Sachse, W and Pao, Y H (1978) On the determination of phase and group velocities of dispersive waves in solids J Appl Phys 49: 4320-4 [11] Mobley, J.; Waters, K R.; Hall, C S.; Marsh, J N.; Hughes, M S.; Brandenburger G.H and Miller, J G (1999) Measurements and predictions of the phase velocity and attenuation coefficient in suspensions of elastic microspheres J Acoust Soc Am 106: 652.-659 [12] Del Gross, V A and Mader, C.W (1972) Speed of sound in pure water J Acoust Soc Am 52: 14421446 [13] Nyquist, H (1932) Regeneration theory Bell System Tech J 11: 126-147 [14] McBumey, T (1992) The relationship between leaf thickness and plant water Potential J Exp Bot 43:327-332 [15] Nakamoto, K.; Oku, T.; Hayakawa, S (1996) Photosynthetic characteristics of tea leaves growth under field conditions Environ Control Biol (in Japanese) 34: 277-283 [16] Fukuhara, M.; Okushima, L.; Matsuo, K and Honma, T (2005) Jpn Agri Res Quart (in press) [17] Fukuhara, M and Sampei, A (1996) Low-temperature elastic moduli and dilational and shear internal friction of polycarbonate Jpn J Appl Phys.35: 3218- 3221 [18] Fukuhara, M.; Kuwano, Y.; Tsugane, A.; Yoshida, M (1999) Determinatin of thermal degradation of volcanized rubbers using diffracted SH ultrasonic waves J Polym Sci Pt B: Polym Phys 37: 497-503 [19] Fukuhara, M and Tsubouchi, T (2003) Naphthenic hydrocarbon oils transmissible for transverse waves Chem Phys Lett 371:184-188 [20] Fukuhara, M.; Yin, F.; Kawahara, K.(2004) Acoustic characteristics of high damping Mn73Cu20Ni5Fe2 alloy Phys Stat Sol.(a) 201: 454-458 [21] Maeda, Y (1957) Dynamic Viscoelasticity, Polymers, In: Lecture on Experimental Chemistry (in Japanese), Vol.8, Maruzen, Tokyo; pp 155 [22] Fukuhara, M.; Kuwano,Y and Oguri, M (1996) Determination of thermal degradation of heated polyvinyl chloride using diffracted SH ultrasonic waves Jpn J Appl Phys 35: 3088-3092 [23] Kuwano, Y.; Fukuhara, M.; Omura, H.; Takayama, S.(1996) Determination of thermal degradation of polypropylene using diffracted SH ultrasonic waves The First Symposium for Polymer Analysis, Jpn Soc Chem Analysis (in Japanese), Inst Nagoya Tech.,Tokyo; pp 155-156 [24] Fukuhara, M and Sampei, A (2000) Ultrasonic elastic properties of steel under tensile stress Jpn J Appl Phys 39: 2916-2921 439 PHYSICAL AND ENGINEERING PERSPECTIVES OF IN VITRO PLANT CRYOPRESERVATION ERICA E BENSON1, JASON JOHNSTON1, JAYANTHI MUTHUSAMY2 AND KEITH HARDING1 Plant Conservation Group, School of Contemporary Science, University of Abertay Dundee, Bell Street, Dundee, DD1 1HG, Scotland, UK- Fax: 00 44 (0) 1382 308261-Email:e.e.benson@abertay.ac.uk Forest Research Institute of Malaysia, Kepong, 52109, Kuala Lumpur, Malaysia Introduction Cryopreservation is the conservation of living cells and organisms at ultra low temperatures usually at -196oC in liquid nitrogen, it is a safe, long-term means of securing in vitro germplasm in culture collections Cryogenic storage is used extensively in medical, horticultural, agricultural, aquaculture and forestry sectors and assists environmental research by preserving test organisms used in environmental monitoring Designing and operating instruments and analytical equipment required to study and conserve biological samples at cryogenic temperatures poses a challenge dictated by the physical and safety constraints of operating at very low temperatures This chapter overviews the physical and engineering aspects of in vitro plant cryopreservation including an introduction to the safe use of cryogenic systems It concludes with a comparative case study of how thermal instrumentation may be applied to develop cryopreservation methods for temperate and tropical plant germplasm maintained in tissue culture Cryobiology is a broad discipline and includes the preservation of a broad spectrum of biodiversity, as well as medical, polar and environmental low temperature research [1] Plant cell cryopreservation requires the input of theoretical and practical expertise from diverse disciplines, including: engineering, computing and physics; chemistry, biology and biotechnology and, increasingly environmental knowledge Cryobiologists network across multidisciplinary sectors, (www.cobra.ac.uk; http://www.sltb.info; http://www.societyforcryobiology.org/; http://www.agr.kuleuven.ac.be/dtp/tro/CRYMC EPT) which historically has lead to the development of generic analytical cryogenic instrumentation and storage technologies [1] Cryogenic engineering is highly specialist, encompassing the design, construction and utilization of equipment capable of effective and safe operations at ultra low temperatures In many situations this involves withstanding the physical constraints of operating, often rapidly, through different thermal cycles, under elevated pressures and in contact with liquid nitrogen vapour and 441 S Dutta Gupta and Y Ibaraki (eds.), Plant Tissue Culture Engineering, 441–476 © 2006 Springer Printed in the Netherlands E.E Benson, J Johnston, J Muthusamy and K Harding liquid Operator and sample safety is an essential component of cryogenic engineering and the provision of specialist protective wear, safety-monitoring instrumentation and procedures must underpin all aspects of its application The properties of liquid nitrogen and cryosafety Nitrogen (N2) makes up ca 80% of the atmosphere; it is odourless, colourless, tasteless, and not detectable by human senses When cooled to its boiling point (-196oC) N2 can be condensed to form Liquid Nitrogen (LN), remaining in this state provided that it is at or below this temperature On warming, N2 is released and a concomitant white vapour frequently and briefly forms containing frozen water Whilst LN is not toxic it has two life-threatening hazards: (a) on evaporation N2 displaces air, creating an atmosphere that cannot support life because of asphyxiation; (b) the severe and extreme cold of LN and its vapours causes serious frostbite and cryogenic burns The safety information provided here is intended as a basic alert for the reader of the potential dangers of handling cryogenic equipment and LN It is the ultimate responsibility of the reader to ensure that full safety assessments are performed and that appropriate protective and safety procedures (including oxygen monitors) are used before handling cryogenic equipment, liquids and facilities As a guide, see the UK’s Health and Safety Executive website (www.hse.gov.uk.) and the suppliers of cryogenic equipment (e.g www.WessingtonCryogenics.co.uk) Together with the other cryogenic gases (helium and argon) very small amounts of LN can evaporate into large quantities of gas in a ratio of about 700:1; in enclosed and limited spaces oxygen becomes depleted and asphyxiation ensues Entry of personnel into atmospheres 40oC) before rapidly cooling in LN and rewarming at 37oC Kartha [11] noted ice formation in this initial study and a high degree of variation in recovery responses, despite this, the method greatly improved upon the application of conventional methods of cryopreservation Benson et al [14] explored Kartha’s original [12] droplet method, but this time by ultra rapidly, cooling naked cassava shoot-tips suspended in 15% (v/v) DMSO droplets of µl, 50 µl and 80 µl sizes Ice nucleation characteristics of the droplets were investigated using visual observations, a temperature probe, and comparisons of ultra rapid and slow cooling (at a rate of -0.5oC min-1) followed by direct exposure to LN Ice nucleation of 5µl and 50µl droplets was not consistently achieved and direct exposure to LN on aluminium foils frequently did not result in the visualization of ice formation (opaque droplets) Suggesting that the droplets on occasions may have formed amorphous ice, whereas the larger 80µl droplets consistently and spontaneously nucleated at ca -22oC This modification [14] of the original Kartha method [12] using the naked freezing of very small cryoprotectant droplets containing meristems was further refined for cryopreserving potato [15] Apices were contained in 2.5µl droplets placed on 0.03mm thick-aluminium foils suspended in cryovials filled with LN However, no details as to the ice nucleation characteristics of the droplet were presented in this study, but as observed in the initial study [14] of cassava it is highly likely that vitrification occurs Grout and Henshaw [13] used hypodermic needles to deliver “naked” potato shoot meristems (in µl-sized droplets of 10% (v/v) DMSO) into LN and postulated the formation of vitreous ice as described by Luyet [16] The “flash-frozen” meristems were only 2-4mm size, so the rapidly conducting needle surfaces may also contribute to the formation of glasses This approach was later successfully applied to cryopreserve shoots of a wider genotype range of potato [17] Wesley-Smith et al [18] developed ultra rapid cooling to cryopreserve recalcitrant plant embryos that cannot tolerate desiccation This explores the possibility that higher cooling rates minimise ice crystallisation, size and growth (Figure 2B and 2D, D3) and thus increases the tolerable water content of cells such that hydrated tissues can withstand cryopreservation This is because at ultra rapid rates of cooling, water molecules cannot energetically arrange themselves into a crystal form Wesley-Smith et al [18] cautions that water mobility is restricted below –134oC and no further ice growth occurs below this temperature However, if hydrated cryopreserved samples are warmed to higher than this critical point, small and unstable ice crystals coalesce, growing larger structures In pure water the critical temperature range for ice formation and growth is to -134oC Cryoprotectants depress the freezing point (generally –30 to –40oC) and increase the temperature of re-crystallization above –134oC, reducing therefore the range of temperatures supportive of ice crystal growth [18] Wesley-Smith et al [19] used the ultra rapid freezing to obtain survival of cryopreserved Camellia sinensis embryos cooled at 200oC min-1 at a water content of 0.7 to 0.4g H2O g-1 dry mass, increasing the cooling rate to 500oC min-1 and 100% survival was reported for higher water contents (1.1 to 1.6 H2O g-1) Technologically this method [18,19] comprises two parts, first, drying germplasm over activated silica gel at 25-28oC to a 449 E.E Benson, J Johnston, J Muthusamy and K Harding critically determined moisture content based on species-desiccation tolerance limits Secondly, cooling ultra rapidly with a specially constructed spring-loaded, rapid plungecooling apparatus [18] devised using cryo-electron microscopy principles The tension and speed of travel of the spring-loaded delivery mechanism into a cryogen (LN/isopentane) delivered cooling rates of 5000-7000oC min-1 Ultra rapid cooling was one of the first approaches used to cryopreserve plants [13,20,21]; its application being superseded, to some extent, by the arrival of controlled rate cooling methods followed by vitrification The potential of using ultra rapid freezing is still however compelling for recalcitrant germplasm as dehydration beyond critical points of desiccation tolerance is not necessary 4.2 CONTROLLED RATE OR SLOW COOLING Mazur’s two-factor hypothesis (Figure 2) explains that the rate of change of temperature at which cells are exposed to freezing controls the rate at which water moves across cell compartments and that this influences cell solute concentration [5] The dynamics of freeze-induced water movement determines survival as water moving from intracellular to extracellular spaces causes a colligative effect as solutes become increasingly concentrated (Figure 2D) During controlled rate freezing ice will initially nucleate extra-cellularly, forming a water vapour deficit that initiates the movement of intracellular water to the outside of the cell whereupon it freezes The process is, in fact cryodehydration and as it progresses the concentration of cellular solutes increase, as a consequence freezing point is depressed and the cells supercool Successful cryopreservation is dependent upon achieving a cooling rate that allows cryodehydration to occur to such an extent that little or no intra-cellular water is available to form ice crystals on exposure to liquid nitrogen (Figure 2D,D1) In the case of cryoprotected cells undergoing slow cooling, water can be supercooled to a temperature of –40oC, the point of homogeneous ice nucleation Applying penetrating “colligative” cryoprotectants to the cell before freezing reduces damaging solution effects of cryodehydration Colligative protection requires cryoprotectants to penetrate the cell (e.g DMSO) and remain in solution at sufficiently low temperatures that allow freezing point depression (supercooling) to a point at which the cell can survive colligative stress Penetrating cryoprotectants act as “cellular solvents”, reducing the concentration of damaging solutes and increasing the unfrozen fraction, thereby limiting the deleterious volume changes Cells cryopreserved by controlled rate cooling are taken to a “terminal transfer temperature” at, or around, the temperature of homogeneous ice nucleation (-40oC) In some cases ice is manually or electronically initiated (“seeded”) at a higher heterogeneous transfer temperature so evoking cryodehydration To ensure that sufficient water has left the cell a holding time is usually incorporated (30-40 minutes) at the terminal transfer temperature, giving the opportunity for more water to be withdrawn After reaching and holding at the terminal transfer temperature cells and cryoprotectants are then immersed in LN Survival ultimately depends on preventing or limiting ice formation in any remaining intra-cellular water, such that: (a) there is not sufficient water to form large ice crystals, those that are formed are so small that they are not injurious or (b) cellular viscosity is so high that any remaining intra-cellular water becomes vitrified 450 Physical and engineering perspectives of in vitro plant cryopreservation 4.3 VITRIFICATION Vitrification is the “solidification” of liquids in the absence of crystallization, a state with a random molecular structure but possessing physical and mechanical properties similar to a solid Glasses are metastable as de-vitrification can occur on re-warming, the glass returning to either a liquid or crystalline state Achieving a stable vitreous state during cryopreservation is important and involves controlling molecular mobility by enhancing cellular viscosity through osmotic, evaporative and cryodehydration and/or by the loading of penetrating cryoprotectants (Figure 2E) High viscosity solutions restrict the ability of H2O molecules to re-arrange into crystals and ice nucleation becomes more difficult as temperature decreases Moreover, the viscosity of highly concentrated solutes rises further during cooling and the molecular mobility of water is virtually arrested At this stage the liquid becomes a glass; the glass transition temperature (Tg) is used to characterize that point at which the physical properties of the system change Glasses; unlike ice crystals not significantly change the structure or composition of solutions and their effects in cryopreservation are less damaging than ice But, cells have to be dehydration and desiccation tolerant because practically the vitrification (Figure 2E) of plant germplasm requires and increase in cell viscosity This is achieved by: evaporative desiccation using still or laminar flow air; chemical desiccants such as silica gel; osmotic dehydration, (sugars and polyols); alginate encapsulation/dehydration and the loading of chemical cryoprotectant cocktails (penetrating and non-penetrating) Vitrification is advantageous as samples are plunged directly into liquid nitrogen but rapid re-warming is critical ensuring movement through the Tg before ice crystallization occurs Vitrification protocols not require controlled rate-cooling apparatus and are “low tech”, whereas programmable freezers are preferred in genebanks so that large accessions can be processed more efficiently Thus, vitrification is best applied to recalcitrant germplasm and laboratories without access to controlled rate freezers Cryoengineering: technology and equipment Cryoengineering is a wide field, mainly developed in medical faculties [22] Plant cryopreservation requires equipment for controlled rate cooling, cryogenic storage and cryogenic shipment this review will also focus on cryomicroscopy and Differential Scanning Calorimetry (DSC) 5.1 CRYOENGINEERING FOR CRYOGENIC STORAGE Mazur presents “The Inverted U” as one of the basic principles of cryopreservation related to the two causal factors intra-cellular and dehydration of cryoinjury [5] These are determined by the rate at which cells are cooled, the “U” describes the graphics of survival versus cooling rate Maximum survival occurs when the dynamics of the excursion of H2O in controlled rate cooling is optimised (Figure 2D) and colligative cryoprotection obviates the damaging effects of excessive solute concentration In plant cryopreservation optimum cooling rates are within the range 0.2 to 1.0oC min-1 for the majority of cell types The precise control of cooling rates, and for many systems, the 451 E.E Benson, J Johnston, J Muthusamy and K Harding temperature at which ice nucleation is initiated is critical to survival and best achieved using specialist apparatus 5.1.1 Controlled rate freezers Withers and King [23] developed one of the first widely applicable, higher plant cell cryopreservation methods using a simple, custom-built controlled rate freezer unit regulated by a solvent coolant The system comprised an insulated plastic bin with a L capacity glass beaker housing a dip cooler capable of chilling to -40oC (temperature of homogeneous ice nucleation) and a heating coil connected to a thermostated bath with a pump to circulate the coolant, 30% (v/v) methanol A temperature probe was inserted for regulation and the samples placed in cryovials and suspended over the coolant in polystyrene rafts Thermocouples were inserted to monitor sample and coolant temperatures The cooling capacity of the freezing unit was calibrated for different volumes of solvent coolant and cooling rates for different volumes determined by thermostatic control of the dip cooler Using this approach the Withers and King [23] devised a slow freezing protocol utilising a 3-component cryoprotectant mixture containing sucrose, glycerol and DMSO and a cooling rate of –1oC min-1 to an intermediate temperature of –35oC at which the cells are placed on hold for 30 minutes before transferring to liquid nitrogen Specifically engineered programmable controlled rate freezers using liquid nitrogen as the coolant were first developed in the 1970s by medical cryobiologist, David Pegg, (now Professor of Cryobiology, University of York, UK) in association with Planer plc, the London-based company who manufactured the equipment (Planer, G personal communications) This system, known as stepwise cooling, was a major breakthrough in human and animal cell cryopreservation and soon became widely applied in medical and animal husbandry sectors Early Planer freezers used a cam controller to produce multi-component cooling regimes and then went on to manufacture full digital computer controllers from the 1980s onwards Today’s Planer units (Figure 3) are based on the principle of a pulse-width modulated solenoid valve to admit liquid nitrogen into the freezing chamber The solenoid pulses on and off at a time varied by the control unit in response to the difference between the actual and desired temperature Planer freezing chambers are typically 16 L capacity and large or smaller units can be manufactured in accordance to fitness of purpose 452 Physical and engineering perspectives of in vitro plant cryopreservation Figure Controlled rate freezer design Freezer units comprise three basic parts: a coolant (LN) delivered by a pressurised dewar system, the freezing chamber that houses the samples and a control unit (Figure 3) Today many companies (Biotronics, Cryomed, Cryologic, L’Air Liquide) have developed controlled rate freezers most of which are based on the original Planer/Pegg design, (Planer, G personal communications) Operations of cryogenic equipment in plant conservation laboratories must take into account functionality in potential extremes of temperature and humidity, particularly in humid, tropical locations Contemporary-designed controlled rate freezers have advantages, as they are robust instruments with respect to environmental parameters They hold reserved liquid nitrogen capacities such that external environmental control is not a limiting factor as they function at relatively low ambient temperature conditions and up to 40oC The main problem however is humidity, which causes door frosting especially in front-loading machines and at delivery port of tubing, an RH limit of 65% is recommended If frosting does occur this causes an accumulation of water so it is important to thoroughly dry the machine as re-cooling causes potential ice damage and the immobilization of samples, moving parts and doors 453 E.E Benson, J Johnston, J Muthusamy and K Harding I High performance storage dewar A Offset neck designed to maintain at -150oC in vapour storage and low liquid nitrogen consumption with standard racks B Durable metal lid for longer life C Rotating interior tray providing easy access to samples D Low maintenance, all stainless steel construction E Annular filling lines designed to reduce frost and ice formation at the lid F Rack stand G.Step up platform (specified by model) II Storage dewar, non-cabinet type A Metal lid designed for longer-life B Tough durable hinges C Annular filling lines designed to reduce frost and ice formation at the lid D High performance under lid temperature E Low maintenance, all stainless steel construction F Tough, durable casters G Vapour platform (optional) Figure Exemplars of high performance and non-cabinet type cryogenic storage dewars (MVE Design), by courtesy of Planer, G and Pattenden, N [Planer Select] Adequate ventilation of LN vapour is required for the venting of waste/exit points from the machine and if the size of the room restricts this then an oxygen monitor with outside repeater alarms and external venting should be in place Programmable freezers are frequently operated with an external dewar (30-40 L), which delivers the coolant LN, via a solenoid valve under pressure to the chamber 454