This action might not be possible to undo. Are you sure you want to continue?
Use of biotechnologies for the conservation of plant biodiversity
Received: 12 April 2010 / Accepted: 14 October 2010 / Editor: P. Lakshmanan # The Society for In Vitro Biology 2010
Abstract In vitro techniques are very useful for conserving plant biodiversity, including (a) genetic resources of recalcitrant seed and vegetatively propagated species, (b) rare and endangered plant species and (c) biotechnology products such as elite genotypes and genetically engineered material. Explants from recalcitrant seed and vegetatively propagated species can be efficiently collected under field conditions using in vitro techniques. In vitro culture techniques ensure the production and rapid multiplication of disease-free material. Medium-term conservation is achieved by reducing growth of plant material, thus increasing intervals between subcultures. For long-term conservation, cryopreservation (liquid nitrogen, −196°C) allows storing plant material without modification or alteration for extended periods, protected from contaminations and with limited maintenance. Slow growth storage protocols are routinely employed for a large number of species, including numerous endangered plants, from temperate and tropical origin. Cryopreservation is well advanced for vegetatively propagated species, and techniques are ready for large-scale experimentation in an increasing number of cases. Research is much less advanced for recalcitrant species due to their seed characteristics, viz., very high sensitivity to desiccation, structural complexity and heterogeneity in terms of developmental
stage and water content at maturity. However, various technical approaches should be explored to develop cryopreservation techniques for a larger number of recalcitrant seed species. A range of analytical techniques are available, which allow understanding physical and biological processes taking place in explants during cryopreservation. These techniques are extremely useful to assist in the development of cryopreservation protocols. In comparison with crop species, only limited research has been performed on cryopreservation of rare and endangered species. Even though routine use of cryopreservation is still limited, an increasing number of examples where cryopreservation is used on a large scale can be found both in genebanks for crops and in botanical gardens for endangered species. Keywords In vitro collecting . Slow growth storage . Cryopreservation . Germplasm conservation . Crops . Rare and endangered species
Introduction A large number of crop species have seeds, which are termed orthodox, i.e. that can be dehydrated down to low water contents and can thus be stored at low temperature for extended periods (Roberts 1973). There are three main categories of plant species for which conservation in seed form is problematic. First, some plants such as banana and plantain do not produce seeds and are thus propagated vegetatively. Second, some species such as potato or sugarcane include both sterile genotypes and genotypes which produce orthodox seeds. However, these seeds are generally highly heterozygous and are thus of limited interest for the conservation of particular genotypes. These species are thus mainly maintained as clones. Third,
F. Engelmann (*) IRD, UMR DIAPC, 911 Avenue Agropolis, BP 64501, 34032 Montpellier cedex 5, France e-mail: firstname.lastname@example.org F. Engelmann Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy
In the case of wild species. the material collected will consist of stakes. Tissue culture techniques are of great interest for the collecting. Bunn et al. For long-term storage. However. conservation of wild. usually that of liquid nitrogen (−196°C). slow growth and cryopreservation techniques are described and analysed in the sections below. a total of 8. It is therefore of paramount importance to develop techniques ensuring optimal storage and rapid multiplication of such species. including numerous rare and endangered species (Fay 1992.and medium-term storage. thus ensuring the production of diseasefree stocks and simplifying quarantine procedures for the international exchange of germplasm. and the number of plants recorded as critically endangered has increased by 60%.321 plant species have been added to the Red List of Threatened Species (IUCN 2004). In vitro propagation protocols have been established for several thousand plant species (George 1996). The development of biotechnology leads to the production of a new category of germplasm including clones obtained from elite genotypes. The miniaturization of explants allows reducing space requirements and consequently labour costs for the maintenance of germplasm collections. all cellular divisions and metabolic processes are stopped. cultures are stored in a small volume. if an adequate sample of the population is to be collected. Botanic gardens and agricultural genebanks should be seen as playing a complementary role for the conservation of plant biodiversity (Engels and Engelmann 1998). In addition. cryopreservation. the world’s biodiversity is declining at an unprecedented rate. 2000). 2006). i. cell lines with special attributes and genetically transformed material (Engelmann 1992). and since the early 1970s. Collectors are faced with various problems when collecting germplasm of recalcitrant seed and vegetatively propagated plant species. as highlighted by Sarasan et al. especially from tropical origin. At this temperature. In vitro collecting. it is not surprising that efforts have been made to improve the quality and security of conservation offered by field genebanks and botanic gardens and to understand and overcome seed recalcitrance to make seed storage more widely available. Collecting missions often require travelling for relatively long periods in remote areas. and they may represent the only option for conserving certain highly endangered and rare species (Ramsay et al. rare and endangered plant species has also become an issue of concern. most activities on ex situ conservation of plant biodiversity have focussed on crop species. corms or suckers. the aim is to reduce growth and to increase the intervals between subcultures.ENGELMANN numerous fruit and forest tree species. This new germplasm is often of high added value and very difficult to produce. produce recalcitrant seeds.org/ourwork/1977/). The development of efficient techniques to ensure its safe conservation is therefore of paramount importance. Moreover. (2006). With vegetatively propagated species. The traditional ex situ conservation method for these categories of plant species is in the form of field collections. Different in vitro conservation methods are employed. is the only current method. However.e. Tissue culture systems allow propagating plant material with high multiplication rates in an aseptic environment. In the light of the problems presented by the different categories of plant species outlined above. it is clear that alternative approaches to genetic conservation are needed for these problem materials. It is thus necessary to keep the material collected in good state for some d/wk before it can be placed in optimal growth or storage conditions. which is a source of problems in terms of volume of material to handle and which induces additional costs. pieces of budwood. Sarasan et al. the traditional conservation approach is in situ conservation. 1991) for which conservation in seed form is still problematic. Botanic gardens play a very important role in ex situ conservation of plant biodiversity. The plant material can thus be stored without alteration or modification for a theoretically unlimited period of time. seeds that cannot be dried to sufficiently low moisture level to allow their storage at low temperature (Roberts 1973). multiplication and storage of plant germplasm (Engelmann 1991. 2007). termed intermediate (Ellis et al. During the period 1996–2004. However. UNEP (1995) estimated that botanic gardens conserve more than one third of the world’s flowering plants. among which Botanic Gardens Conservation International identified more than 15. i. it is now recognised that ex situ techniques can be efficiently used to complement in situ methods.bgci. depending on the storage duration requested. protected from contamination. which limit its efficacy and threaten the safety of plant genetic resources conserved in this way. Not only will most of these explants not be adapted to survival once excised from the . specifically in vitro or tissue cultures. many recalcitrant seeds have a sheer weight and bulk. There is also a large number of species. There are thus great risks that recalcitrant seeds either germinate or deteriorate before they are brought back to the genebank or botanic garden. storage at ultra-low temperature. Indeed. Virus-free plants can be obtained through meristem culture in combination with thermotherapy. requiring very limited maintenance. attention has turned to the possibilities offered by biotechnology. Until now. 1990.e. tubers. For short. Applications of Biotechnologies for Conservation In vitro collecting.000 threatened species (http://www. Conservation in the field presents major drawbacks.
reduction of sugar concentration. continuous inputs are required and long-term questions remain as regards the genetic stability of the stored material. crop plants. which depend on the cold sensitivity of the species. its volume and the volume as well as the type of closure of the culture vessel (Engelmann 1991). shoot tips. 2002a. transported and grown successfully if placed under adequate conditions. b). its implementation still needs customizing to any new material. Even with careful planning of the time of the collecting mission. Musa in vitro plants can be stored at 15°C without transfer for up to 15 mo (Banerjee and De Langhe 1985). The critical points to consider for the development of in vitro collecting techniques have been synthesized and analysed by Withers (1995). Classical techniques involve freeze-induced dehydration. a storage experiment performed with an in vitro collection of African coffee germplasm including 21 diversity groups revealed a large variability in the response of the diversity groups to the storage conditions (Dussert et al. most of the experimental systems employed in cryopreservation (cell suspensions. Moreover. Slow Growth Storage Growth reduction is generally achieved by modifying the environmental conditions and/or the culture medium. Vitrification can be defined as the transition of water directly . including Cryopreservation Techniques Some materials. However. These problems can be overcome if it is realized that the seed is not the only material which can be collected: Zygotic embryos or vegetative tissues such as pieces of budwood. 1984). yam. Some groups showed high genetic erosion during storage whilst others did not show any erosion. changes in the nature and/or concentration of growth regulators and addition of osmotically active compounds. 1997a). Cryopreservation Cryopreservation is the only technique currently available to ensure the safe and cost-efficient long-term conservation of the germplasm of problem species. 2006). as well as wild and endangered species (Pence et al. As an example. it is not always possible to apply one single protocol for conserving genetically diverse material. Razdan and Cocking 1997. both from temperate and tropical origin. shed from the plant or eaten by grazing animals (Guarino et al. whereas new techniques are based on vitrification. summarize the achievements made and problems faced with vegetatively propagated and recalcitrant species and present the current utilization of cryopreservation for plant material. which provide guidance to researchers and genebank and botanic garden managers for the establishment and management of in vitro germplasm collections. which can be combined with a decrease in light intensity or culture in the dark. potato. Difficulties can also be encountered when collecting germplasm of orthodox seedproducing species. Other tropical species such as cassava are much more cold-sensitive since cassava shoot cultures have to be conserved at temperatures higher than 20°C (Roca et al. Sarasan et al. there might be no or little seed available for all or part of the germplasm to be collected. e. Numerous parameters influence the efficiency of in vitro slow growth storage protocols including the type of explants. In this section. if in vitro conservation appears as a simple and practical option for long-term conservation of numerous species and has obvious wide medium-term applications. such as orthodox seeds or dormant buds. Tropical species are often cold-sensitive and have to be stored at higher temperatures. simple and efficient in vitro collecting techniques have been developed for different materials including embryos or vegetative tissues of various species including crops such as coconut or cacao. 2004). their physiological state when entering storage. apices or even leaf discs can be sampled. the type of culture vessel. 1995). Cells have thus to be dehydrated artificially to protect them from the damages caused by the crystallization of intracellular water into ice (Mazur 1984). However. Modifications of the culture medium can include dilution of mineral elements. The most widely applied technique is temperature reduction. or seeds might not be at the optimal developmental stage.g. In vitro slow growth storage techniques are being routinely used for medium-term conservation of numerous species. display natural dehydration processes and can be cryopreserved without any pretreatment. embryos) contain high amounts of cellular water and are thus extremely sensitive to freezing injury since most of them are not inherently freezing tolerant. The techniques employed and the physical mechanisms upon which they are based are different in classical and new cryopreservation techniques (Withers and Engelmann 1998). Technical guidelines have been published recently (Reed et al. Engelmann 1999) and rare and endangered species (Fay 1992. Musa. calluses. shoots. cassava (Ashmore 1997.BIOTECHNOLOGIES FOR CONSERVING BIODIVERSITY parent plant but they will also present health risks due to their vegetative nature and contamination with soil-borne pathogens (Withers 1987). we briefly describe the various cryopreservation techniques available. Following an expert meeting organised by IBPGR in 1984 and sponsorship of various research programmes.
embryos). an increasing amount of the extracellular solution is converted into ice. followed by ice formation in the medium (Mazur 1984). requiring only minor modifications for different cell types (Engelmann 1997b). However. (c) encapsulation–vitrification. Therefore. their use can be avoided by performing the slow-freezing step with a domestic or laboratory freezer (Kartha and Engelmann 1994). Dereuddre et al. cryoprotection. By precluding ice formation in the system. and the cells remain unfrozen but supercooled. Classical cryopreservation techniques have been successfully applied to undifferentiated culture systems such as cell suspensions and calluses (Kartha and Engelmann 1994. each with unique requirements under conditions of freezeinduced dehydration.g. too intense freeze-induced dehydration can incur different damaging events due to concentration of intracellular salts and changes in the cell membrane (Meryman et al. (d) dehydration. rapid immersion of samples in liquid nitrogen. the cells and the external medium initially supercool. Rewarming should be as rapid as possible to avoid the phenomenon of recrystallization in which ice melts and reforms at a thermodynamically favourable. rapid cooling and rewarming.0°C/min) to a determined prefreezing temperature (usually around −40°C). dehydration with highly concentrated vitrification solutions. The encapsulation–dehydration procedure is based on the technology developed for the production of artificial seeds. Vitrification involves treatment of samples with cryoprotective substances. whilst avoiding the formation of crystalline ice (Fahy et al. 1990. in which samples are encapsulated in alginate beads. pregrown in liquid medium enriched with sucrose for 1 to 7 d. vitrification-based procedures are operationally less complex than classical ones (e. In optimal conditions.ENGELMANN from the liquid phase into an amorphous phase or glass. which contain a variety of cell types. In vitrification-based procedures. Engelmann et al. 1992). With temperature reduction during slow cooling. As a result. (e) pregrowth. 2008). Encapsulation–vitrification is a combination of encapsulation–dehydration and vitrification procedures. most or all intracellular freezable water is removed. Vitrification-based procedures offer practical advantages in comparison to classical freezing techniques. removal of cryoprotectants and recovery. Survival is high and growth recovery of cryopreserved samples is generally rapid and direct. different amounts of water will leave the cell before the intracellular contents solidify. rapid thawing and recovery. As the temperature is further decreased. if samples to be frozen are amenable to desiccation down to sufficiently low water contents (which vary depending on the procedure employed and the type and characteristics of the propagule to be frozen) with no or little decrease in survival in comparison to non-dehydrated controls. In some cases. Classical cryopreservation techniques. Classical cryopreservation techniques involve slow cooling down to a defined prefreezing temperature. The cell membrane acts as a physical barrier and prevents the ice from seeding the cell interior. A common feature to all these new protocols is that the critical step to achieve survival is the dehydration step and not the freezing step. Seven different vitrification-based procedures can be identified: (a) encapsulation–dehydration. 1984). as in classical protocols. Withers and Engelmann 1998) and apices of cold-tolerant species (Reed and Uchendu 2008). 1991.5–2. Explants are encapsulated in alginate beads. thus reducing or avoiding detrimental intracellular ice formation upon subsequent immersion of the specimen in liquid nitrogen. cells equilibrate by loss of water to external ice. This is followed by rapid cooling. (f) pregrowth–dehydration and (g) droplet–vitrification. (b) a procedure actually termed vitrification. then frozen rapidly. Classical techniques are generally operationally complex since they require the use of sophisticated and expensive programmable freezers. then subjected . they do not require the use of controlled freezers) and have greater potential for broad applicability. all factors that affect intracellular ice formation are avoided. they are more appropriate for complex organs (shoot tips. 2008). without callus formation. Depending upon the rate of cooling and the prefreezing temperature. Like ultra-rapid freezing. cell suspensions and somatic of numerous different species (Sakai and Engelmann 2007. New cryopreservation techniques. cell dehydration is performed prior to freezing by exposure of samples to concentrated cryoprotective media and/or air desiccation. followed by rapid immersion in liquid nitrogen. partially desiccated in the air current of a laminar air flow cabinet or with silica gel to a water content around 20% (fresh weight basis). Since cells remain supercooled and their aqueous vapour pressure exceeds that of the frozen external compartment. 1977). Sakai et al. This procedure has been developed for apices. larger and more damaging crystal size (Mazur 1984). no or limited further drop in survival is generally observed after cryopreservation (Engelmann 1997b). thus resulting in the concentration of intracellular solutes. Niino et al. Classical freezing procedures include the following successive steps: pregrowth of samples. This technique has been applied to apices of numerous species from temperate and of tropical origin as well as to cell suspensions and somatic embryos of several species (Gonzalez-Arnao and Engelmann 2006. Glass transitions (changes in the structural conformation of the glass) during cooling and rewarming have been recorded with various materials using thermal analysis (Sakai et al. slow cooling (0. storage.
including in vitro micropropagation. Many vegetatively propagated species successfully cryopreserved until now are cultivated crops. The meristematic zone of apices. 2002). dehydrated under the laminar airflow cabinet or with silica gel and then frozen rapidly. different protocols can be employed for a given species and produce comparable results. Optimal survival is generally obtained when samples are frozen with a water content comprised between 10% and 20% (fresh weight basis). 1993). Reed 2008). For vegetatively propagated species. and pregrowth procedures and relatively homogenous samples in terms of size. then placed on an aluminium foil in minute droplets of vitrification solution and frozen rapidly in liquid nitrogen. and callusing only or transitory callusing is often observed before organised regrowth starts. The whole meristem is generally preserved when vitrification-based techniques are employed. Other reasons for the good results obtained are linked with tissue culture protocols. whatever their storage characteristics. cryopreservation has a wide applicability both in terms of species coverage. 1990. Pence 1995. cellular composition. In view of the wide range of efficient and operationally simple techniques available. b). In addition. and callusing is observed only in cases where the technique is not optimized. b. ornamentals and plantation crops. A number of publications provide lists of species which have been successfully cryopreserved (Engelmann 1997a. then freezing them rapidly by direct immersion in liquid nitrogen. Droplet–vitrification is the latest technique developed (Sakai and Engelmann 2007). thus reducing desiccation injury (Berjak et al. It has been applied to embryos of a large number of recalcitrant and intermediate species (Engelmann 1997a). often of great commercial importance. since protocols have been successfully established for root and tubers. This method has been applied notably to asparagus stem segments. Engelmann and Takagi 2000. classical procedures often lead to destruction of large zones of the meristems. physiological state and growth response are employed for freezing. Engelmann et al. no ice formation takes place in vitrification-based procedures. fruit trees. Ultra-rapid drying in a stream of compressed dry air (a process called flash drying developed by Berjak’s group in South Africa) allows freezing samples with a relatively high water content. with little vacuoles and a high nucleocytoplasmic ratio. vitrification-based procedures allow using samples of relatively large size (shoot tips of 0. In a pregrowth–dehydration procedure. 1995. for which cultural practices. organised regrowth. The pregrowth technique has been developed for Musa meristematic cultures (Panis et al. With a few exceptions. oil palm somatic embryos and coconut zygotic embryos (Uragami et al. However. This technique is mainly used with zygotic embryos or embryonic axes extracted from seeds. Engelmann 1997a. is composed of a relatively homogenous population of small. Recalcitrant seed species.5 to 2–3 mm).BIOTECHNOLOGIES FOR CONSERVING BIODIVERSITY to freezing by vitrification. Survival is generally high to very high. This might lead to the conclusion that freezing of embryos is a routine procedure applicable to numerous species. Allium. careful examination of the species . Dumet et al. explants are pregrown in the presence of cryoprotectants. It is also interesting to note that in many cases. thus increasing the chances of positive and uniform response to treatments. Sakai et al. are well established. thus avoiding the extensive damage caused by ice crystals which are formed during classical procedures. It has been applied to apices of an increasing number of species (Sakai and Engelmann 2007. provided that the tissue culture protocol is sufficiently operational for this species. 1989). Cryopreservation of Vegetatively Propagated and Recalcitrant Seed Species Vegetatively propagated species. Apices are pretreated with vitrification solution. Some publications present extensive lists of plant species whose embryos and/or embryonic axes have been successfully cryopreserved (Kartha and Engelmann 1994. As mentioned earlier. e. then freezing them rapidly by direct immersion in liquid nitrogen. Assy-Bah and Engelmann 1992. Cryopreservation techniques are now operational for large-scale experimentation in an increasing number of cases. Different reasons can be mentioned to explain these positive results. 2008). which can regrow directly without any difficulty. Regeneration is rapid and direct. thus allowing direct. The pregrowth technique consists of cultivating samples in the presence of cryoprotectants. vitrification-based protocols have been employed. both from temperate and tropical origin and in terms of numbers of genotypes/varieties within a given species. By contrast. yam and potato. Desiccation is usually performed in the air current of a laminar airflow cabinet. Finally. in vitro material is “synchronized” by the tissue culture. and up to 100% survival could be achieved in some cases. The number of species to which it has been successfully applied is increasing steadily. actively dividing cells. These characteristics make them more susceptible to withstand desiccation than highly vacuolated and differentiated cells. any vegetatively propagated species should be amenable to cryopreservation. but more precise and reproducible dehydration conditions are achieved by using a flow of sterile compressed air or silica gel. from which organised growth originates.g. Dehydration is the simplest procedure since it consists of dehydrating explants.
Some species previously classified as recalcitrant have thus been moved to the intermediate or even suborthodox categories and stored using classical or new storage techniques (Engelmann 2000). A classical protocol. in these cases. There is scope for technical improvements in the current cryopreservation protocols for embryos and embryonic axes. involving treatment with DMSO followed by slow cooling. including pregrowth–desiccation. However. has also been very effective for cryopreservation of several species (Berjak et al. Survival is extremely variable. There are various options to consider for improving storage of non-orthodox seeds. This might be the only solution for species which do not have well-defined embryos. as well as for bryophytes and ferns (Pence 2008) using the techniques described in the above sections. Lambardi et al. which seldom have been employed so far with recalcitrant species. The desiccation technique is mainly employed for freezing embryos and embryonic axes (Normah and Makeen 2008). this will require that more sophisticated tissue culture procedures be developed and mastered. Pregrowth on media containing cryoprotective substances may confer the tissues’ increased tolerance to further desiccation and to reduce the heterogeneity of the material. Desiccation has been employed for freezing seeds of rare temperate orchids by direct immersion in liquid nitrogen (Nikishina et al. 1997b). 1995). However. however. 2007. Finally. encapsulation– dehydration and vitrification. 2009). tissue culture protocols. germination and growth of plants. research is still at a very preliminary stage for recalcitrant seeds. the root pole seeming more resistant than the shoot pole. very precisely controlled desiccation (e. basic protocols for disinfection. Engelmann 2000). including inoculation in vitro. and they are wild species in their large majority. germination of embryos or embryonic axes. This is because research in this area is recent and addressed by very few teams worldwide and because recalcitrance is a dynamic concept which evolves with research on the biology of species and improvement in classical storage procedures. and embryos or embryonic axes are thus successfully employed for cryopreservation. Seeds of many species are of too large dimensions to be frozen directly. regeneration frequently restricted to callusing or incomplete development of plants and the number of accessions tested per species generally very low. Encapsulation–dehydration has been used notably for cryopreserving protocorms of Celisostoma areitinum. ancient and wild Citrus species (Malik and Chaudhury 2006. it has been suggested to use shoot apices sampled on the embryos. Finally. Other cryopreservation techniques. little or nothing is known on the biology and all the more so on the seed storage behaviour of many of these species. In some species. Wesley-Smith et al. First of all. In comparison to the results obtained with vegetatively propagated species. 2007). a rare Thai orchid (Maneerattanarungroj et . Cryopreservation of Rare and Endangered Species The number of publications on cryopreservation of rare and endangered species is still relatively limited. With species for which attempts to freeze whole embryos or embryonic axes have proven unsuccessful. was successful for freezing seeds of 68 native Western Australian species out of the 90 species tested (Touchell and Dixon 1993). Seeds and embryos of recalcitrant species also display very important variations in moisture content and maturity stage between provenances. freezing protocols have been developed for various higher plants (Bunn et al. as observed notably with cacao (Chandel et al. embryos are often of very complex tissue composition which displays differential sensitivity to desiccation and freezing. are often non-existent or not fully operational. there is a huge number of species with recalcitrant or suspectedly recalcitrant seeds. In cases where some information on seed storage behaviour is available. using saturated salt solutions) and cooling conditions may allow to freeze whole seeds. as demonstrated recently with various coffee species (Dussert et al. With some species. inoculation in vitro. should be experimented on (Pence 1995. embryos of some species are too large to envisage using them for cryopreservation.ENGELMANN mentioned in these papers reveals that only a limited number of truly recalcitrant seed species is in fact included. adventitious buds or somatic embryos induced from the embryonic tissues (Pence 1995). selecting embryos at the right developmental stage is of critical importance for the success of any cryopreservation experiment (Engelmann et al.g. as well as between successive harvests. As a consequence. plant development and possibly limited propagation will have to be established prior to any cryopreservation experiment. 1989. There are a number of reasons to explain the current limited development of cryopreservation for recalcitrant seed species (Engelmann 2000). 2006). which make their cryopreservation difficult. and even minor reduction in their moisture content—down to levels much too high to obtain survival after freezing—leads to irreparable structural damage. Several authors have used the desiccation technique for freezing seeds of endangered. Hamilton et al. 2007). which were applied to different explant types. between and among seed lots. followed by ultra-rapid freezing. including orchids (Hirano et al. Flash drying. rare. embryos are extremely sensitive to desiccation. propagation and acclimatization which are needed for regrowth of embryos and embryonic axes after freezing. and seeds of some species do not contain well-defined embryos. 1995). 1992). However.
see also http://www. using apices sampled from in vitro plants. Niino 1995). Golmirzaie and Panta 2000). In China. Corvallis. 2005. Peru. 2000).bgpa. 2006) and more than 200 accessions at the International Potato Center (Lima. (2009) report successful cryopreservation of shoot tips of endangered Australian and Japanese species. More than 1. 2007) and of wild relatives of Diospyros (Niu et al.au/). including collections of seeds of regional endangered species (Pence 1991). using a protocol including after controlled dehydration and freezing (Dussert and Engelmann 2006). 2009). Germany at the Leibnitz Institute of Plant Genetics and Crop Plant Research (Keller et al. France. Cryopreservation is also applied to biotechnology products. Turner et al. cryopreservation is used mainly for storing seeds with limited longevity and of rare or endangered species. pollen of more than 700 accessions of traditional Chinese flower species is conserved under cryopreservation (Li et al.org/earth/CREW/frozengarden. which are. the NBPGR conserves cryopreserved pollen of 65 accessions belonging to different species (Mandal 2000). as is the case for the 420 accessions of the mulberry field collection maintained at the National Institute of Agrobiological Resources (Yamagata.cincinnatizoo.000 callus strains of species of pharmaceutical interest are stored at −196°C in the UK (Spencer 1999). The plant for which its development is the most advanced is potato. 1999) and the banana lines produced in Vitropic. 2004).BIOTECHNOLOGIES FOR CONSERVING BIODIVERSITY al.html). consisting mainly of endangered medicinal plants (Mandal 2000). personal communication). In France. cryopreservation is systematically employed for storing all the new embryogenic cell lines of coffee and cacao produced by the Biotechnology Laboratory of the Nestlé Company located in Notre Dame d’Oé. In France. In the USA. cryopreservation is being applied in genebanks for long-term storage of genetic resources of vegetatively propagated species. Dormant buds of more than 440 European elm accessions are conserved in liquid nitrogen by Afocel (Bordeaux Nangis. France. some of which have been stored for more than 15 yr (Ganeshan and Rajashekaran 2000).200 accessions from 50 different species. 1999). 2007). which is an interesting material for genetic resource conservation of various species. Embryogenic cultures of around 80 oil palm accessions have been cryopreserved and stored at IRD (Dumet 1994). Cañas.000 old potato varieties are cryostored in Gatersleben. New Delhi. respectively. there is a growing number of genebanks and botanic gardens where cryopreservation is employed on a large scale for different types of materials. 2009). Large-Scale Utilization of Cryopreservation for Germplasm Conservation Even though its routine use is still limited. India) stores 1. A duplicate of around 100 accessions of the Pyrus field collection National Clonal Germplasm Repository (NCGR. CO) conserves 43. The National Center for Genetic Resources Preservation (NCGRP. (2001) and Tanaka et al. two cryopreserved collections of Allium have been established.400 accessions over the vapours of liquid nitrogen (Walters 2010. Fort Collins. the 2. as well as several thousand conifer embryogenic cell lines employed in large-scale clonal planting programmes in Canada (Cyr 2000). France). Cryopreserved collections of coffee seeds are being established in Tropical Agricultural Research and Higher Education Center (CATIE. and research is under way in France (IRD) and the USA (NCGRP) for grape germplasm conservation. since more than 1. in the framework of a national grape genetic resources conservation project. using the vitrification technique. seeds of several hundred accessions are being cryopreserved after partial desiccation (Dussert 2010. Breeders routinely store pollen in liquid nitrogen in the framework of their improvement programmes (Towill and Walters 2000). Japan. gov. In the USA. Harvengt et al. . Finally. In India. Finally. In the case of orthodox seed species. is stored with this aim by several institutes. 2000). a private tissue-culture laboratory based in Saint-Mathieu de Tréviers. This technique is also used in several botanic gardens. Costa Rica) and in IRD (Montpellier. The National Bureau for Plant Genetic Resources (NBPGR. Cryopreservation is also applied to intermediate seeds which are tolerant to freezing. which comprise a total of more than 800 accessions (Kim et al. and shoot tips of the endemic endangered plant Centaurium rigualii (Gonzalez-Benito and Perez 1997). 2009).200 accessions of the US apple germplasm field collection are duplicated under cryopreservation (Forsline et al. The droplet–vitrification technique has been used for freezing shoot tips of wild potatoes (Yoon et al. In Korea. tolerant to dehydration. the NCGRP conserves pollen of 13 pear cultivars and 24 Pyrus species (Reed et al. of endangered plant tissues and of spores and tissues of Bryophytes and Pterydophytes (http:// www. and the Indian Institute for Horticultural Research (Bangalore.wa. India) conserves pollen of 600 accessions belonging to 40 species from 15 different families. More than 110 accessions of rare or threatened species are stored under cryopreservation at the Kings Park and Botanic Garden in Perth. Pollen. or are not. with another duplicate at the NCGRP (Reed et al. personal communication). Guanacaste. France (Florin et al. the Center for Conservation and Research of Endangered Wildlife at the Cincinnati Zoo and Botanical Garden conserves several cryopreserved collections. In the case of dormant buds. OR) is cryostored at NCGR. Australia (Touchell and Dixon 1994.
grapevine virus A. chromosomal or molecular level which could be attributed to cryopreservation (Engelmann 1997b. the lower the per accession cost. raspberry (Rubus idaeus). a detailed study compared the costs of maintaining one of the world’s largest coffee field collections with those of establishing a coffee cryocollection at CATIE in Costa Rica (Dulloo et al. 2007) and 540 cassava accessions at the International Center for Tropical Agriculture (CIAT. 2007). Another one is their broad applicability. i.000 under in vitro slow growth storage. US $23 under in vitro slow growth storage and only US $1 under cryopreservation. 2008). hundreds or even thousands in the cryobank. plant pathogens such as viruses. An important advantage of these new techniques is their operational simplicity. Cryopreservation imposes a series of stresses to the plant material. To date. cryopreservation may be the method of choice for long-term conservation of genetic diversity. There is no report of modifications at the phenotypical. More recently. to which US $50–60 should be added once for cryopreserving this accession. Additional Uses of Cryopreservation Recently. grapevine (Vitis vinifera). which is of particular relevance to conservation of wild species. as a substitute or in complement to classical virus eradication techniques such as meristem culture and cryotherapy (Wang et al. raspberry bushy dwarf virus. Prunus spp. Belgium (Panis et al. and the number of cases where it is used routinely is increasing steadily. Roca (personal communication) evaluates the annual maintenance cost of CIAT’s cassava collection. Gonzalez-Arnao et al. potato (Mix-Wagner et al. probabilistic tools have been developed recently to assist genebank curators in the establishment and management of cryopreserved germplasm collections (Dussert et al. 2003). Uneven distribution of viruses and obligate vasculature-limited microbes in shoot tips allows elimination of the infected cells by injuring them with the cryotreatment and regeneration of healthy shoots from the surviving pathogen-free meristematic cells. cucumber mosaic virus. 2009).000 under cryopreservation. cryopreservation techniques are sufficiently advanced to envisage their immediate utilization for large-scale application. against US $30. plum pox virus. potato virus Y. potato leaf roll virus. for which large amounts of genetic diversity need to be conserved. biochemical. Cryopreservation: Progress and Prospects Even though cryopreservation is still routinely employed in a limited number of cases. which requires the establishment of specific procedures for their management. In addition to cost. which are susceptible of inducing modifications in cryopreserved cultures and regenerated plants. a sweet potato little leaf phytoplasma and a bacterium. The few studies performed on the cost of cryopreservation confirm the interest of this technique from a financial perspective. phytoplasmas and bacteria are eradicated from shoot tips by exposing them briefly to liquid nitrogen. the study examined the advantages of cryopreservation over field collection and showed that for species that are difficult to conserve using seeds and that can only be conserved as live plants. which includes 5. Colombia. at around US $5.e. Thermotherapy followed by cryotherapy of shoot tips can be used to enhance virus eradication. since they will be mainly applied in developing tropical countries where the largest part of genetic resources of problem species is located. 2003). 2004). the development of the new vitrification-based freezing techniques has made its application to a broad range of species possible (Engelmann 2004).. for eliminating viruses from infected plants. It is thus necessary to verify that the genetic stability of the cryopreserved material is not altered before routinely using this technique for the long-term conservation of plant genetic resources. at the NCGR. The large-scale utilization of cryopreservation implies a scaling up of material to handle and to store from one or a few genotypes in the laboratory to several tens. severe pathogens in banana (Musa spp. Research is much less advanced for .. Cali. Citrus spp. cryopreservation has been used for cryotherapy.).ENGELMANN cryopreserved collections are under development for longterm storage of tropical plants: 630 banana accessions have been cryopreserved at the INIBAP International Transit Center. It allows treatment of large numbers of samples and results in a high frequency of pathogen-free regenerants. The results indicate that cryopreservation costs less (in perpetuity per accession) than conservation in field genebanks. These pathogens include nine viruses (banana streak virus. in view of its low utilization cost. sweet potato feathery mottle virus and sweet potato chlorotic stunt virus). Difficulties related to excision and regeneration of small meristems are largely circumvented. Hummer and Reed (2000) indicate that. In cryotherapy. Leuven. 2000) did not reveal any differences in the characters studied. potato (Solanum tuberosum) and sweet potato (Ipomoea batatas) have been eradicated using cryotherapy. sugarcane (Gonzalez-Arnao 1996) and banana (Côte et al. Recent studies comparing the vegetative and floral development in the field of plants originating from control and cryopreserved material performed with several species including oil palm (Konan et al. In this aim. Cryotherapy of shoot tips is easy to implement. Huanglongbing. For many vegetatively propagated species.000 accessions. A comparative analysis of the costs of both methods showed that the more accessions there are in cryopreservation storage. the annual cost of one temperate fruit tree accession is US $77 in the field. 2008).
Rome. 1992. References Ashmore S.. Panis B. Conclusion In this paper.. the development of appropriate complementary conservation strategies will still require further research to define the criteria. Plant Cell Rep 4: 351–354. Dereuddre J.htm.. As already mentioned in this paper. CryoLetters 12: 135–148... research is actively performed by various groups in universities. botanic gardens and genebanks worldwide to improve knowledge of biological mechanisms underlying seed recalcitrance. especially in the area of cryopreservation. This Action aims notably at improving fundamental knowledge about cryoprotection through the determination of physicobiochemical changes associated with tolerance towards cryopreservation and at developing and applying new cryopreservation protocols. Vasquez N. Thermal analysis. R. Snook L. our understanding of the biological mechanisms involved in cryopreservation will increase and that cryopreservation will become more frequently employed for the long-term conservation of plant genetic resources. De Langhe E. It is hoped that new findings on critical issues such as understanding and control of desiccation sensitivity will contribute significantly to the development of improved cryopreservation techniques for recalcitrant seed species. Planta 186: 249–261..kuleuven. They offer genebank and botanic garden curators additional tools to allow them to improve the conservation of germplasm collections placed under their responsibility. J. 1991. JIRCAS. Eira M. Dulloo M. Bunn E. Malik S. K. 1997.. 2007. Mycock D.. Farrant J. Tsukuba. Dussert S. as well as the cost-effectiveness and security afforded by their application. Qamar Z. Ebert A. 2000. However.. Bellachew B. A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions of Musa (banana and plantain). Selection of the appropriate methods should be based on a range of criteria.. research institutes. Rakotomalala J. dramatic progress has been made with the development of new conservation techniques for non-orthodox and vegetatively propagated species. Anthony F. Chandel K. with very different characteristics.. Banerjee N. CryoLetters 13: 117–126. there are various technical approaches to explore to improve the efficiency and to increase the applicability of cryopreservation techniques to recalcitrant species. and feasibility of the particular methods chosen (which depend on the availability of the necessary infrastructures)..) to desiccation and freezing in liquid nitrogen: 2. be/dtp/tro/cost871/Home. 1995. including the biological nature of the species in question. Engelmann F.. S...... and the current ex situ conservation concepts should be modified accordingly to accommodate these technological advances. Domergue R. W.. Hassen M.. 1989. E. X. pp 261–268. Aust J Bot 55: 345–355. it is interesting to mention that an EU-funded COST (European Cooperation in the field of Scientific and Technical Research) Action (COST 871: Cryopreservation of crop species in Europe) has been initiated recently. Chaudhury R. R. Astorga C. M. In many instances. W. CryoLetters 21: 19–24. Cyr D. the complementarity of genebank and botanic conservation should be fully recognised and capitalized upon to optimize plant biodiversity conservation. Watts J. Assy-Bah B. Cryopreservation: roles in clonal propagation and germplasm conservation of conifers. Pammenter N. Homoiohydrous (recalcitrant) seeds: the enigma of their desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer. 2009. During recent years. see http://www. refine the methods and test their application for a range of genepools and situations. Radhamani J.. Goue O. It is now well recognised that an appropriate conservation strategy for a particular plant genepool requires a holistic approach. Considerations of complementarity with respect to the efficiency and cost-effectiveness of the various conservation methods chosen are also important. In situ and ex situ methods.. Côte F. Blandin S. practicality . Gotor E. are options available for the different genepool elements.biw. 2000.) obtained after regeneration of cryopreserved embryogenic cell suspensions. it is important to stress that the new.. It can thus be realistically expected that in the coming years. 1985. Crop Sci 49: 2123–2138. W. The contribution of in vitro technology and cryogenic storage to conservation of indigenous plants.. In addition. we have presented the new possibilities provided by biotechnologies for improving ex situ conservation of plant biodiversity in genebanks and botanic gardens.. cocoa and jackfruit. Jenny C.... J. Cryopreservation of mature embryos of coconut (Cocos nucifera L. combining the different ex situ and in situ conservation techniques available in a complementary manner. For additional information. Kaminski M. Ann Bot 76: 443–450. Omondi C.. Resistance of alginate-coated somatic embryos of carrot (Daucus carota L. In this context. Engelmann F. Status report on the development and application of in vitro techniques for the conservation and use of plant genetic resources. Dixon K. Takagi H.. Panaia M. which fall within this category and to the comparatively limited level of research activities aiming at improving the conservation of these species. This is due to the large number of mainly wild species. Rabemiafara A. P. Cost efficiency of cryopreservation as a long term conservation method for coffee genetic resources.. Berjak P.BIOTECHNOLOGIES FOR CONSERVING BIODIVERSITY recalcitrant seed species. (eds) Cryopreservation of tropical plant germplasm—current research progress and applications. In this regard. Desiccation and freezing sensitivity in recalcitrant seeds of tea. efficient in vitro conservation techniques developed are not seen as replacements for conventional ex situ approaches. Turner S.. In-field behavior of banana plants (Musa AA sp. International Plant Genetic Resources Institute. including a range of techniques for the latter... In: Engelmann F.) and subsequent regeneration of plantlets.
1994. Grübe M. Brulard E. Tesis presentada en opcion al grado de Doctor en ciencias tecnicas. pp 195–230. Duval Y. R. 1992.. Dordrecht. T. Florin B. Engelmann F. F. Fifth international botanic gardens conservation congress. New determinants of coffee (Coffea arabica L.. Chabrillange N. Centro de Bioplantas... (ed) Plant cryopreservation: a practical guide. In: Engelmann F. Mii M. Kirstenbosch. E. Anthony F. R. (eds) Cryopreservation of tropical plant germplasm—current research progress and applications. In: da Silva JA Teixeira (ed) Floriculture. South Africa. Dereuddre J.. Cryopreservation of coffee (Coffea arabica L. In vitro conservation of tropical plant germplasm—a review. Lamboy W. Cryopreservation in the Gatersleben genebank—state of the art in potato. Engelmann F. Development of probabilistic tools to assist in the establishment and management of cryopreserved plant germplasm collections. 1984. Biodivers Conserv 6: 583–590. pp 281–290. Takagi H.. Hong T.. An intermediate category of seed storage behaviour? I. Engelmann F. Current status of pollen cryopreservation research: relevance to tropical agriculture. II. garrawayi. CABI. Development and large scale application of cryopreservation techniques for shoot and somatic embryo cultures of tropical crops. Cali. Acta Hort 484: 75–78.. Anthony F. CIAT. Engels J. McFerson J. 1999. Gallais A. Engelmann F. 167. 2006. 2000. Colombia. Leunufna S. Rome. Dussert S. Botanic gardens and agricultural genebanks: building on complementary strengths for more effective global conservation of plant genetic resources.. Reed B. M. Wu W.. JIRCAS. Slow growth storage and cryopreservation—tools to facilitate germplasm . 2004. In: Vasil I. J. Rao R. Reid R. M.) seed tolerance to liquid nitrogen exposure. Development of encapsulation–dehydration.. Gonzalez-Arnao M. N. Springer.. IUCN Red List of Threatened Species. Tsukuba. Coffee. Ashmore S. Cryopreservation of oil palm (Elaeis guineensis Jacq. 1997.) seeds: importance of the precooling temperature. thesis. 2008. V. JIRCAS. CIAT. Takagi H. Euphytica 57: 227–243. MacFarlane D. Senula A. E. In: Engelmann F. 1996.. (eds) Cryopreservation of tropical plant germplasm—current research progress and applications. Wallingford... vol. 1992. Advances in potato cryopreservation at the International Potato Center. Dumet D.. 1994 Dumet D. 1998.. Cryoconservation des massifs d’embryons somatiques de palmier à huile (Elaeis guineensis Jacq. Seed storage behaviour in Elaeis guineensis. Plant Genet Resour Newsl 112: 9–18. Duval Y. J Exp Bot 41: 1167–1174. Guarino L. Engelmann F. Establishment and operation of a temperate clonal field genebank. Grübe M. Plant Genet Resour Newsl 103: 27–31. Advances in orchid cryopreservation. CryoLetters 27: 169–178. Factors affecting the cryopreservation of coffee.D. Engelmann F. (ed) Management of field and in vitro germplasm collections. pp 29–31. Hummer K.ENGELMANN Dumet D. Tsukuba. France.. Rome. Gonzalez-Arnao M.. Berlin. Hamon S. Cryobiology 21: 407–426. Dussert S. Plant propagation by tissue culture. Pritchard H. Chabrillange N. H. Kartha K. Etude du rôle du saccharose pendant le prétraitement. 1995. 1993.. Development of base and active collections of Malus germplasm with cryopreserved dormant buds. Engelmann F.. Congresso Internacional Biotecnología y Agricultura (Bioveg 2005).. 1997a. 1999... CryoLetters 30: 268–279. Callow J.. Thorpe T. CryoLetters 27: 155–168. Dussert S. Exegetics. inodora and C. Cali. pp 360–365.. Plant Cell Rep 16: 344–348. Cryopreservation of plant germplasm using the encapsulation–dehydration technique: review and case study on sugarcane.. T.. In Vitro Cell Dev Biol Plant 40: 427–433. 1995.. Plant Cell Tiss Org Cult 92: 1–13. K. Senula A. Chabrillange N. Angell C.... Seed Sci Res 1: 99–104. E. France. 2003. International Plant Genetic Resources Institute.. 2000. M. Peru. Edington. Ellis R. 1997a. 2004. Abdelnour-Esquivel A.. technical guidelines. H. garlic and mint. Ellis R. Fay M. World Congress of the Society for Cryobiology. Meryman H. La Habana. Meier-Dinkel A.) core collection under slow growth conditions. Conservation of rare and endangered plants using in vitro methods. W. Springer. Roberts E. Hong T. www. Desarollo de una tecnica para la crioconservacion de meristemos apicales de caña de azucar.. Centro Nacional de Investigaciones Cientificas. Keller E. E. Proceedings of a consultation meeting—15–20 January.. ornamental and plant biotechnology.. Tsukuba. 1997b. Assy-Bah B. Golmirzaie A. Engelmann F. In: Engelmann F.... M. Engelmann F. Université Paris 6. In: Engelmann F. 1999. R.. 1991. R. Variability in storage response within a coffee (Coffea spp. Fahy G. Tsukuba. Cuba. pp 410–414. Vitrification as an approach to cryopreservation. Cryopreservation and germplasm storage. CryoLetters 18: 269–276. Ganeshan S. Establishment of a cryopreserved gene bank of European elms. Importance of cryopreservation for the conservation of plant genetic resources.. 2005. Ishikawa K. J. 14–18 September.. Thermal analysis and cryopreservation of seeds of Australian wild Citrus species (Rutaceae): Citrus australasica. Escobar R. Gonzalez-Benito M. Global Science Books... Roberts E. In: Abstracts Cryo’99. Wellingford. Gonzalez-Arnao M... In: Ford-Lloyd B. pp 8–20. In Vitro Cell Dev Biol Plant 28: 1–4. Cryopreservation of embryos. Establishment of a cryopreserved coffee germplasm bank.. pp 119–162... E.. 2009. H. 2008. Rajashekaran P. Importance of desiccation for the cryopreservation of recalcitrant seed and vegetatively propagated species. Berlin. H. Plant Cell Rep 12: 352–355.. International Plant Genetic Resources Institute. Hamon S. T. Proceedings of a consultation meeting—15–20 January 1996. Kluwer. JIRCAS. (eds) Reproductive biology and plant breeding.. Collin E. Engelmann F. Paris. Management of field and in vitro germplasm collections. 2000. Part 2—in practice. Cryopreservation of tropical plant germplasm—current research progress and applications. Proc. Recalt C. Plant cryopreservation: progress and prospects. In: Dattée Y... Cuba. In vitro conservation methods. Engelmann F. George E. Towill L. Engelmann F. Marseilles. Colombia..) par déshydratationvitrification. Ikenobe. advances and topical issues. Cryopreservation of nodal explants of an endangered plant species (Centaurium rigualii Esteve) using the encapsulation–dehydration method. L. Harvengt L.. T. A. 1991... Panta A..org. K. 2006. (eds) Cryopreservation of tropical plant germplasm—current research progress and applications.. Takagi H. 1997b. E. pp 59–76. M. In: Reed B. Ciego de Avila. IUCN. E... Hirano T. Collecting plant genetic diversity.. 12–15 July.. F. Engelmann F. Hamilton K. Dumas E.. pp 250– 254. Escobar R. Engelmann F. T. Engelmann F. coconut and oil palm embryos. Noirot M. CryoLetters 24: 149–160.. Dumas C. 2006. 1996 Gonzalez-Arnao M. 2000. Engelmann F.) somatic embryos involving a desiccation step. Lepage B. Ph.. Panta A. A. Engelmann F. Chabrillange N. A. 2004. Can J Forest Res 34: 43–55. 2000. (eds) Plant cell and tissue culture. 1996. J. Roca W. Engelmann F. H. Engelmann F.. (eds) Biotechnology and plant genetic resources: conservation and use.iucnredlist. 2nd ed. Keller E. CAB International. Dussert S. F. Mem. Newburry J. M.. Forsline P. JIRCAS. C. Perez C.. Soetisna U. 1990. Takagi H.
Russ J Plant Physiol 54: 121–127. Rome. Cryopreservation of shoot tips of endangered Hayachine-usuyukiso (Leontopodium hayachinense (Takeda) Hara et Kitam. I. M. M. Benelli C. Plant Genet Res: Charact Utiliz 4: 204–209. S. H. Reed B. C. Application of cryopreservation protocols at a clonal genebank. Wang H. 1993.. Ann Bot 40: 541–546. Nikishina T. (eds) Cryopreservation of tropical plant germplasm—current research progress and applications.. In: Reed B.. 1991. R. Strosse H. Koadio Y. Reed B. M. Takagi H. Liu Y. 2007. 1990.) Garay.. In: Engelmann F.. D. Pence V.... Annual meeting of the Society for Cryobiology. In: Pence V. 2000. Walters C. Shumilov V... Cryopreservation of recalcitrant seeds. 1995. F. Predicting the viability of seeds. D. Dulloo M. 441–446.. M. 1977. Enfield. Reyes R. Bunnag S. Tsukuba. Niamké A. Las Palmas de Gran Canaria. M. Pollen cryo-bank establishment and application of traditional Chinese flowers. application and validation of plant cryopreservation. Uemura M.. Marseilles.) using a vitrification protocol. Springer. M. W. Makeen A. Yakuwa H. Williams R. sixth symposium of the international society for tropical root crops. Plant cryopreservation: a practical guide. P. Italy. Niino T.. Cocking E. Cryopreservation of in vitrogrown shoot tips of apple and pear by vitrification. Leuven. Burov A. L. Malik S. Kim H. J. Halmagyi A. Berlin. P. Springer. Takagi H. Recovery of potato apices after several years of storage in liquid nitrogen. Freezing of living cells: mechanisms and applications.. Razdan M. 245.... Tsukuba. Meryman H.. encapsulation–vitrification and droplet–vitrification: a review. Engelmann F. Cryopreservation of garlic germplasm collections using the droplet–vitrification technique. Sandoval J. S. Luo Z. Plant Cell Rep 9: 30–33. Yu V. K. Engelmann F. 1984. Cryopreservation of bryophytes and ferns. C. Cryopreservation of plant germplasm I. B.. 2006. 21–26 February.. Takagi H. McMichen M. Prendergast G. The challenges of developing cryopreservation strategies to suit the requirements of a large industrial in vitro plant cell collection. (ed) Plant cryopreservation: a practical guide. pp 117–140. M. In: Reed B.. Engels J. Beltran J. 1st Meeting of COST 871 Working Group 2: Technology.. Ramsay M.. Cryopreservation of germplasm of mulberry. Touchell D. f. var. 2008. Sandoval J. Cryopreservation of plant germplasm I. Swennen R.... S.. Handbook for Genebanks N 7. 12–15 July. Kolomeitseva G. In: Proc. IPGRI technical bulletin N7. pp 211–240. In: Engelmann F. 10–13 May. Tsukuba. K. Porley R. Schumacher H. Carlo A. Asian J Plant Sci 6: 1235–1240. Li B. K. 2000.. Technical guidelines for the management of field and in vitro germplasm collections. Rome. 1973. In vitro conservation of Cleisostoma areitinum (Rchb. rare Thai orchid species by an encapsulation–dehydration method. Biodivers Conserv 2: 594–602.. Atherton C. J Wuhan Bot Res 27: 451–454. CryoLetters 23: 375–384. In: Abstracts Cryo’99.. Niino T.. In vitro collecting techniques for germplasm conservation. Sakai A. C. Seed cryopreservation for conservation of ancient Citrus germplasm. H. Berlin. Cryobiology 14: 287–302. Conservation in vitro of threatened plants—progress in the past decade. 5–8 April.. Tsuchiya Y. DeNoma J. Popova E. Wild and endangered species.. 2008.. In: Abst. Cripps R. Swennen R.. Cryopreservation of seed of Western Australia native species. Berlin. M. . In: BGCI (ed) Eurogard 2000—II European botanic garden congress. 2003. Mix-Wagner G... Spencer M... M. The cryopreservation of embryonic axes of two wild and endangered Citrus species.. In: Abst.. Effect of various factors on minimal growth in tissue culture storage of cassava germplasm. Zhang Y. Maneerattanarungroj P. No N. Springer. W. Reed B. Dumet D. Panis B. CryoLetters 24: 33–41. pp 102–113. L. Dixon K. Pence V. Niino T. Berlin... Adv Hortic Sci 21: 198–202. Int Refrig J 29: 411–417. 1984. Song C. Yoon M.. Studies on cryopreservation of two Diospyros spp. Pence V.. Normah M. Berlin. Monthatong M. pp 52–57. E. M. 1997. 2002a. Uchendu E. pp 33–58. Y. Rowntree J.. 2006... S. Chaudhury R. Rome. 2006. Volume 1: general aspects. (eds) In vitro collecting techniques for germplasm conservation. C. S. J. K. 34–35... Seed Sci Technol 19: 235–251. Springer. Shirokov A. Peru. Dixon K. Vitrification. L. Van den Houwe I.. pp 76–82. 2009. C. C. J. M. IPGRI technical bulletin N 7. Panis B. germplasm by modified droplet vitrification. Development of PVS-based vitrification and encapsulation–vitrification protocols. (ed) Biotechnology in agriculture and forestry vol 32.. Jackson A. Clark J. Cryopreservation of excised embryos and embryonic axes. Popovich E. Cryopreservation of the banana germplasm collection at the ITC (INIBAP Transit Centre).. Villalobos V. Sakai A. pp 77– 92. 108. C... pp 246–249. M. Zhang Y.. (eds) Cryopreservation of tropical plant germplasm—current research progress and applications... Roberts H. IPGRI. 2009. S. Mazur P. France. Roca W. Duval Y. pp 115–129. Tsukuba. Lima. 1992. M.. Sakai A.. Plant Genet Res Charact Util 6: 164–166. CryoLetters 28: 151–172.. V. Engelmann F. In: Engelmann F. 2007. JIRCAS.. Springer. C. Reed B.. 1994. pp 29–52. L. N. Cross R.. Amer J Physiol 247(Cell Physiol 16): C125–C142... J... Kobayashi S. 1st international symposium on cryopreservation in horticultural species.. 2009.. F. Rival A.. H. 2000. Springer. 1995. Sarasan V. 21–26 July. Pence V. 2007. In: Bajaj Y. 2002b. CRYO ’09.. 2007. Springer. Berlin. Cryopreservation of seeds of Ohio native plants and related species. Florence. Cryopreservation research in India: current status and future perspectives. E. Ramsay M. (ed) Biotechnology in agriculture and forestry vol 32. V. Pence V... G.. Science. Cryopreservation of seeds and protocorms of rare temperate orchids.. 2007.. Oiyama I. Plair B. Lambardi M. Niino T. Vakhrameeva M. Belgium. In Vitro Cell Dev Biol—Plant 42: 206–214. JIRCAS. Konan E. In: Bajaj Y. CryoLetters 28: 377– 386. JIRCAS. Engelmann F. Freezing injury from solution effects and its prevention by natural or artificial cryoprotection. Lee J.. R. Cryopreservation for seedbanking of Australian species. Hirai D. Villalobos V. Tanaka D. IPGRI/SGRP.. Engelmann F. In: Reed B. (ed) Plant cryopreservation: a practical guide. Choi H.. 2004. Engelmann F. Shirata K. Varlygina T.. Japan.) originating from cryopreserved stabilized polyembryonic cultures (SPCs). 2002.. K. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. 2008. (ed) Plant cryopreservation: a practical guide. L. Piette B. IPGRI. J. 2008.. Niu Y. Touchell D. de Vettori C. Douglas M. Plant Cell Tiss Org Cult 28: 261–266. Cryopreservation of pollen.BIOTECHNOLOGIES FOR CONSERVING BIODIVERSITY maintenance of vegetatively propagated crops in living plant collections. Durand-Gasselin T. (eds) Cryopreservation of tropical plant germplasm—current research progress and applications. In: Reed B.. Conservation of plant genetic resources in vitro. In: Proc. Controlled rate cooling. M. Sakai A. 2000.. 2007. pp 282–286. 2009. Van den Henda S. 1999.. T. Towill L. 2008. A pilot study for the ex situ conservation of UK bryophytes.. Mandal B. Field development of oil palms (Elaeis guineensis Jacq. A. Popov A. EBGC.. M. H. I. V. Nojiri K. 39. Shin D. brasiliensis Tanaka) by vitrification. World Congress of Cryobiology.. Sucrose preculture to simplify cryopreservation of banana meristem cultures. Berlin.. Chang Y. (ed) Plant cryopreservation: a practical guide. Seed Sci Technol 1: 499–514.
1992. T.. A.. Panis B. 1995.interscience.. In vitro conservation of plant genetic resources. Kim H.. Senaratna T.ENGELMANN Turner S. Ko H. Cryopreservation of shoot tips from six endangered Australian species using a modified vitrification protocol... W. C. A. W. (eds) Collecting plant genetic diversity. E. Withers L. Engelmann F.. 1995. Ann Appl Biol 154: http://www3. freezing rate and the thermal properties of tissue water.wiley. G. 1998. S. H. In vitro methods for germplasm collecting in the field. Wang Q. Wallingford. In: Altman A.. Cho E... Engelmann F. Elimination of plant pathogens by cryotherapy of shoot tips: a review. Cambridge University Press. Bunn E. CAB International. 1987. Reid R. W. Vertucci C. Cambridge. Tan B. Berjak P. J Plant Physiol 140: 596– 604. Hwang H. Acta Hortic 760: 203–208. Lambardi M. Global biodiversity assessment. New York. W. Cryopreservation of cultivated and wild potato varieties by droplet vitrification procedure. Withers L. H. Marcel Dekker. FAO/IBPGR Plant Genet Resour Newsl 69: 2–6..... Withers L. Plant Cell Rep 9: 328–331.. Sakai A. Touchell D. Engelmann F. Pammenter N. . Crane J.. 1990. grown in vitro. 2001. Collecting in vitro for genetic resources conservation. Wesley-Smith J.. Uragami A. 2007. 2008. Yoon J.. Magai M. Park Y. Valkonen J.. pp 511–515. A. UNEP.. Ann Bot 87: 371– 378. P..com/ journal/119879031/issue. R.. (ed) Biotechnology in agriculture. Dixon K. Rao R. pp 57–88. In: Guarino L. Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L... Cryopreservation of desiccation-sensitive axes of Camellia sinensis in relation to dehydration.
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue reading from where you left off, or restart the preview.