<Emphasis Type="Italic">In vitro</Emphasis> conservation of Malaysian biodiversity—achievements,challenges and future directions

Malaysia is fortunate and proud to contain some of the world’s richest biodiversity. In Malaysia, there are an estimated 185,000 species of fauna and 12,500 species of flowering plants, many of which are endemic to tropical forests in this region. Indeed, such diversity is an important and invaluable national asset to safeguard both present and future generations. In vitro conservation offers possible techniques for the preservation of plant germplasm that at present is difficult to maintain or is maintained with limited success. Research at the Universiti Kebangsaan Malaysia (The National University of Malaysia) focuses on the cryopreservation of woody fruit species with seeds that cannot tolerate cryopreservation (recalcitrant or intermediate). Among the plants with recalcitrant seeds are such traditionally important edible tropical fruits as mangosteen, langsat, and rambai (Garcinia mangostana, Lansium domesticum, and Baccaurea motleyana). Citrus aurantifolia, Citrus suhuiensis, Citrus madurensis, Citrus hystrix, and Fortunella polyandra are among the Citrus and Citrus-related species studied. Cryopreservation studies include the Nepenthes species (pitcher plants) of Malaysia. Fundamental research on desiccation and low-temperature tolerance and on the physiology of desiccation are used to understand seed behavior, a prerequisite for the development of successful conservation techniques. At the same time, cryopreservation protocols for several Citrus and forestry species were developed for embryonic axes and adventitious shoots, mainly using rapid dehydration and PVS2 vitrification techniques. There are no successful standard techniques or protocols for species with highly recalcitrant seeds such as Garcinia species. Modification of existing protocols or development of new methods is required, but this can be accomplished only when a detailed understanding of the recalcitrant nature of the seeds or explants is achieved. While we have considerable knowledge concerning the basics of biochemical processes and some molecular data from work on desiccation-tolerant seeds, a great need remains for understanding the cause of the recalcitrance or desiccation sensitivity of these seeds. It may be necessary to use a systems biology approach that exploits the “omics” technologies to generate global molecular data. In combination with bioinformatics for data integration and analyses, this approach would move toward improved modeling of the biological pathways associated with the development of recalcitrant seeds.     

Importance of <Emphasis Type="Italic">in vitro</Emphasis> technology to future conservation programmes worldwide

The latest IUCN statistics show that of over 12,000 plant species, 70% are threatened, 19% are critically endangered and 28 species are extinct in the wild. Target 8 of the Global Strategy for Plant Conservation (GSPC) highlights the importance of ex situ conservation of critically endangered plants. Long-term germplasm storage for species with recalcitrant seeds needs alternative measures. In vitro methods complement seed banking and other ex situ measures and are vital for long-term conservation. Conservation Biotechnology at RBG Kew is currently working on a number of rare and threatened recalcitrant species from biodiversity-rich areas of the world to develop good quality in vitro propagules for cryopreservation, recovery and restoration projects. The importance of successful in vitro propagation methods, transplantation technologies, cryopreservation and international networking for the integrated conservation of these species are discussed in detail.     

Plant cryopreservation: Progress and prospects

Summary Cryopreservation (liquid nitrogen, −196°C) represents the only safe and cost-effective option for long-term conservation of germplasm of non-orthodox seed species, vegetatively propagated species, and of biotechnology products. Classical cryopreservation techniques, which are based on freeze-induced dehydration, are mainly employed for freezing undifferentiated cultures and apices of cold-tolerant species. New cryopreservation techniques, which are based on vitrification of internal solutes, are successfully employed with all explant types, including cells suspensions and calluses, apices, and somatic and zygotic embryos of temperate and tropical species. The development of cryopreservation protocols is significantly more advanced for vegetatively propagated species than for recalcitrant seed species. Even though its routine use is still limited, there are a growing number of examples where cryopreservation is employed on a large scale for different types of materials, including seeds with orthodox and intermediate storage behaviour, dormant buds, pollen, biotechnology products, and apices sampled from in vitro plantlets of vegetatively propagated species. Cryopreservation can also be employed for uses other than germplasm conservation, such as cryoselection, i.e., the selection through freezing of samples with special properties, or cryotherapy, i.e., the elimination of viruses from infected plants through apex cryopreservation. Because of its high potential, it is expected that cryopreservation will become more frequently employed for long-term conservation of plant genetic resources.     

Use of biotechnologies for the conservation of plant biodiversity

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.     

A study of some biochemical and histopathological responses of wet-stored recalcitrant seeds of Avicennia marina infected by Fusarium moniliforme

Although fungi cause a recognized problem during storage of recalcitrant seeds of many tropical species, there are no data to date on defence strategies of these seeds against fungal attack. To ascertain whether recalcitrant seeds of Avicennia marina elaborate compounds that might suppress fungal proliferation during hydrated storage, the production and efficacy of beta-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) were studied in relation to histopathological changes. Freshly harvested seeds had low beta-1,3-glucanase and chitinase activities and fluorescence microscopy revealed progressive deterioration of the internal tissues of these seeds associated with fungal infection during hydrated storage. In seeds treated to minimize associated fungi (clean seeds), beta-1,3-glucanase and chitinase activities increased significantly during 10 d of hydrated storage. Similar high levels of activity were observed when these seeds were experimentally infected with Fusarium moniliforme and subjected to further storage. The histopathological observations indicated delayed disease development in the 10-d clean-storage period, although the hypersensitive response was not observed. The results suggest that, although the recalcitrant seeds of A. marina elaborate some antifungal enzymes, there is a lack of effective defence strategies that might lead to successful responses against fungal infections.     

低温保存技术在顽拗性种子种质保存中的利用

由于顽拗性种子不耐脱水且对低温敏感,常规保存方法难以达到长期保存的目的。因此,(超)低温保存顽拗性种子种质是最理想的方法。顽拗性种子的低温保存,应用较多的是玻璃化法和两步法。诸多因素影响着低温保存的成败,如种子或胚的含水量水平、溶液低温保护剂效应、降温冰冻与解冻方式、水合过程以及后培养等,这些需深入探索与解决。除顽拗性种子脱水耐性和低温敏感性机理外,植物细胞的冻害和抗冻机理也亟需探明,以便找到最佳冷冻方法,制定长期保存种质基因的最佳方案。     

The possibilities and challenges of <Emphasis Type="Italic">in vitro</Emphasis> methods for plant conservation

Seed-based methods are generally the most efficient for propagating and storing plant germplasm, but these methods are not always adequate, and some species can benefit from in vitro methods for conservation. For species that produce few or no seeds in the wild, plants may be propagated in vitro, and in vitro shoot tips can provide material for cryostorage when seeds are not available or are recalcitrant. In vitro propagated plants may also serve as subjects for research, without depleting the genetic resources of the species. Clonal plants can be used to test out suitable habitat and can be used for basic research on endangered species, without disturbing the wild population. Despite the effectiveness of widely used techniques, however, there are still species that resist initiation into culture or that may be difficult to root or acclimatise. Similarly, tissue cryopreservation methods may be restrained by cost, particularly in maintaining multiple genotypes of many species. Maintaining such genotypes in vitro is also costly and runs the risk of loss or change over time. Examples of the successful use of in vitro methods will illustrate the variety of applications of these techniques, but costs and specific challenges will also be discussed to help define areas where further research is needed to realise the potential of in vitro methods as a tool for conservation.     

Development and large scale application of cryopreservation techniques for shoot and somatic embryo cultures of tropical crops

Shoot-tips and somatic embryos are the explants of choice for the in vitro long-term storage of ex situ plant genetic resources in liquid nitrogen. Cryopreservation of organized structures has significantly progressed, especially for species of tropical origin, with the development of several vitrification-based procedures such as encapsulation-dehydration, vitrification and droplet-vitrification approaches. They have allowed improvements in survival and recovery after cryopreservation compared with conventional crystallization-based protocols, proving their effectiveness for large scale application with embryos and shoot-tips of different plants. This review addresses the main physical and technological aspects involved in plant cryopreservation methods, illustrating the development of research with three cases: citrus, cassava and potato. These studies demonstrate how cryopreservation strategies are increasingly applied for their successful employment in the genebanks.     

Orthodox Behaviour of Oil Palm Seed and Cryopreservation of the Excised Embryo for Genetic Conservation

Desiccation experiments have shown the oil palm seed to be orthodox,not recalcitrant, in character. There is a significant differencein water content between the whole seed and the embryo whichis maintained despite desiccation, and results in the failureof sub-zero storage of whole seeds. The successful recoveryof viable, excised embryos from liquid nitrogen, and their subsequentregrowth in vitro, suggests a practical technique for the longterm conservation of the genetic resources of the species. Elaesis guinensis L, oil palm embryo, cryopreservation, recalcitrant seed     

The use of plant stress biomarkers in assessing the effects of desiccation in zygotic embryos from recalcitrant seeds: challenges and considerations

Zygotic embryos from recalcitrant seeds are sensitive to desiccation. In spite of their sensitivity, rapid partial dehydration is necessary for their successful cryopreservation. However, dehydration to water contents (WCs) that preclude lethal ice crystal formation during cooling and rewarming generally leads to desiccation damage. This study investigated the effects of rapid dehydration on selected stress biomarkers (electrolyte leakage, respiratory competence, rate of protein synthesis, superoxide production, lipid peroxidation, antioxidant activity and degree of cellular vacuolation) in zygotic embryos of four recalcitrant‐seeded species. Most biomarkers indicated differences in the levels of stress/damage incurred by embryos dried to WCs < and >0.4 g·g^?1, within species; however, these changes were often unrelated to viability and percentage water loss when data for the four species were pooled for regression analyses. Dehydration‐induced electrolyte leakage was, however, positively related with percentage water loss, while biomarkers of cellular vacuolation were positively related with both percentage water loss and viability. This suggests that electrolyte leakage and degree of cellular vacuolation can be used to quantify dehydration‐induced stress/damage. Biomarkers such as superoxide production, whilst useful in establishing the nature of the dehydration stress incurred may not be able to distinguish the effects of different WCs/drying times. Irrespective of which biomarker is used, the data suggest that understanding differences in desiccation sensitivity across recalcitrant‐seeded species will remain a challenge unless these biomarkers are related to a generic desiccation stress index that integrates the effects of percentage water loss and drying time.     

Evaluating costs for the <Emphasis Type="Italic">in vitro</Emphasis> propagation and preservation of endangered plants

In vitro methods provide opportunities for propagating and preserving endangered plant species when seed-based methods are not adequate. Such species include those that produce few or no seeds, as well as species with recalcitrant seeds. Tissue culture propagation methods can be used to produce such plants for reintroduction, research, education, display, and commerce. They can also be the basis for tissue banking as a way to preserve genetic diversity when seeds cannot be banked. With some recalcitrant species, embryo banking, a method which also utilizes in vitro culture for recovery germination, is possible. The number of endangered species that will require in vitro methods is estimated to be at least 5,000 worldwide. Further information is needed to identify these species, and the ongoing collection of information into databases on endangered species and recalcitrant species will help provide this. The costs of these methods are higher than for traditional propagation and preservation, but they may be necessary for species under higher threat. The multiplication rate of a culture, as well as the rates of rooting and acclimatization, has a major effect on the number of transfers needed for producing plants or tissue for banking, and improvements that will increase the efficiency of these steps can help lower costs. Further research into factors affecting the growth of tissues in vitro, as well as coordination of efforts among institutions with infrastructure for in vitro work, should facilitate the application of in vitro methods to the endangered species that cannot be propagated or preserved using seeds.     

黄皮种子线粒体呼吸速率和活性氧清除酶活性对脱水的响应及其生态学意义

顽拗性种子脱落时具有较高的含水量和代谢活性, 对脱水高度敏感; 但顽拗性种子脱水敏感性的机理至今仍然不清楚。该文以顽拗性黄皮(Clausena lansium)种子为材料, 研究了种子和胚轴对水分丧失的响应, 在脱水过程中胚轴和子叶的呼吸速率, 胚轴和子叶线粒体的细胞色素c氧化酶(CCO)活性、外膜完整性、CCO和交替氧化酶(AOX)途径以及线粒体活性氧清除酶活性的变化。结果表明, 随着水分的丧失, 种子和胚轴的存活率逐渐下降, 种子的脱水敏感性大于胚轴; 胚轴和子叶的呼吸速率以及线粒体外膜的完整性降低。胚轴和子叶线粒体的CCO途径以及胚轴AOX途径的呼吸速率在脱水初期增加, 随着继续脱水下降, 胚轴线粒体AOX途径的呼吸速率则随着脱水显著下降。胚轴线粒体的超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性和子叶线粒体的APX活性随着脱水迅速下降; 胚轴线粒体的脱氢抗坏血酸还原酶(DHAR)活性和子叶线粒体的SOD、DHAR和GR活性在脱水初期增加, 然后下降。这些数据表明黄皮种子的脱水敏感性与线粒体的呼吸速率和活性氧清除酶的活性降低密切相关, 也与长期适应热带/亚热带的生境有关。     

Dehydration in the cryopreservation of moist plant tissues and in seed maturation

The possibility of long-term cryopreservation of plant objects depends on their water content. In orthodox seeds, it decreases at the late stage of maturation and is accompanied by the synthesis of protectors--sugars and proteins. These seeds easily withstand cryopreservation. Organs with a high water content, meristems, and recalcitrant seeds are dried in presence of sucrose before plunging in liquid nitrogen. In orthodox seeds, artificially dried moist seeds, and meristems, the cellular content forms glass structures that are estimated in frozen materials by differential scanning calorimetry and electron paramagnetic resonance methods. It is proposed that the glass cellular content is connected with the duration of cryopreservation. Methodical approaches to successive cryopreservation of moist plant tissues are described.     

植物种质资源超低温保存概述

简要回顾了植物种质资源超低温保存的历史,说明了超低温保存植物材料的多样性,阐述了超低温耐性的生物学基础及超低温伤害产生的原因和类型,介绍了各种常用超低温保存方法的技术要点,并对生产顽拗性种子的植物种质资源的超低温保存作了专门的论述,分析了生产顽拗性种子的植物种质资源超低温保存的潜力、现状和困难,指出顽拗性种子的超低温保存是植物种质资源超低温保存的重点和难点,而真正实现用超低温保存技术贮藏顽拗性植物种质资源还有很长的路要走。     

Desiccation sensitivity and successful cryopreservation of oil seeds of European hazelnut (Corylus avellana)

The sensitivity of a Polish provenance of dormant Corylus avellana seeds to extreme desiccation and cryopreservation in liquid nitrogen (LN, ?196°C) was investigated. No sensitivity to desiccation was observed as seeds readily germinated and exhibited seedling emergence, even when the critical water content (WC) of seeds (nuts devoid of pericarp) was reduced to 0.027 g H₂O/g dry mass, g g^?1 by drying. Results of germination and seedling emergence tests indicated that seeds tolerated cooling in LN when desiccated to a WC in the range of 0.05–0.10 and 0.08–0.10 g g^?1, respectively. The results of this study demonstrate the feasibility of long‐term cryopreservation of European hazelnut seeds. As the seeds of this species have been classified in several different categories (orthodox, suborthodox and recalcitrant), based on their response to desiccation and low temperature, the assignment of the seeds of hazelnut to a specific category is provided and discussed.     

Conservation <Emphasis Type="BoldItalic">in vitro</Emphasis> of rare and threatened ferns—case studies of biodiversity hotspot and island species

The importance of in vitro tools to complement other ex situ methods for saving plants from extinction is more relevant than ever before. More than 50% of the world’s plant species are endemic to the 34 global biodiversity hotspots (GBHs), each holding at least 1,500 endemic plant species. In addition, a large number of small islands hold a number of endemic species on the brink of extinction. Conservation support concentrating more on these hotspots and small islands would significantly reduce the loss of species that is currently occurring. In the majority of these cases, the resources are either locally scarce or difficult to access for in vitro conservation to support other ex situ measures. Most island countries are small, and their geographical position is a stumbling block to initiate active partnerships with other countries when they need to use in vitro tools to rescue plants that produce recalcitrant seeds/spores or propagate only by vegetative means. However, many biodiversity hotspot countries have facilities and expertise, and they concentrate on their own flora for in vitro conservation programmes. For decades, because of the grave threat these plants face, the Conservation Biotechnology Unit, previously known as the Micropropagation Unit, at Royal Botanic Gardens Kew (RBG Kew) has been at the forefront of assisting countries to save their valuable biodiversity through both in situ and ex situ methods. Approaches mentioned here highlight work on recalcitrant ferns from GBHs and small islands. Source materials from recalcitrant species, either spore or seed and in some cases vegetative material, need to be used immediately after collection for tangible results in vitro. This becomes more difficult when only a few plants or small populations are left in the wild. The task becomes harder when available material is small in quantity, and there is greater restriction on the use of available genetic diversity in the wild. This paper highlights the importance of proper collection measures, in vitro culture procedures and cryopreservation and methods for the integrated conservation of threatened ferns from both GBHs and small islands. The importance of international networking to achieve these conservation goals also will be discussed.     

Detection of dehydrin-like proteins in embryos and endosperm of mature Euterpe edulis seeds

Summary. Euterpe edulis Martius, a tropical palm species characterized as highly recalcitrant, accumulated dehydrin proteins in both the endosperm and the embryo of the mature seed, as detected by Western blot analysis and immunogold electron microscopy. Three major bands at molecular masses of approximately 16, 18, and 24 kDa were identified in both samples analysed. Immunogold electron microscopy studies detected the presence of dehydrins in the embryo and endosperm. In both cases, dehydrins were immunolocalized in cytoplasm and chromatin. No labelling associated with either membranes or organelles was detected. It is known that dehydrins are produced as part of the developmental program of orthodox seeds and are also present in some recalcitrant seeds of temperate regions. The constitutive presence of dehydrins in embryos of extremely recalcitrant species of tropical origin has not been previously reported. Correspondence and reprints: Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EHA, Ciudad Autónoma de Buenos Aires, Argentina.     

DNA methylation of Quercus robur L. plumules following cryo-pretreatment and cryopreservation

Pedunculate oak (Quercus robur) is an ecologically and economically important forest tree species which produces seeds that are classified as recalcitrant. Thus, cryopreservation of seed meristems is a method for long-term preservation of this germplasm in gene banks. During cryopreservation, many factors, such as desiccation, cryoprotection and cooling/rewarming, can induce stress in the frozen meristems. In this study, in vitro survival and the global DNA methylation level of plumules after cryoprotection, desiccation and cryostorage was evaluated. Results indicated that both desiccation and storage in liquid nitrogen have negligible influence on DNA methylation status of Q. robur plumules. These findings support the cryopreservation of plumules as an appropriate method for conservation of Q. robur germplasm.     

Carbon monoxide enhances the chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated glutathione homeostasis

Both carbon monoxide (CO) and nitric oxide (NO) play fundamental roles in plant responses to environmental stress. Glutathione (GSH) homeostasis through the glutathione-ascorbate cycle regulates the cellular redox status and protects the plant from damage due to reactive oxygen species (ROS) or reactive nitrogen species (RNS). Most recalcitrant seeds are sensitive to chilling stress, but the roles of and cross talk among CO, NO, ROS, and GSH in recalcitrant seeds under low temperature are not well understood. Here, we report that the germination of recalcitrant Baccaurea ramiflora seeds shows sensitivity to chilling stress, but application of exogenous CO or NO markedly increased GSH accumulation, enhanced the activities of antioxidant enzymes involved in the glutathione-ascorbate cycle, decreased the content of H(2)O(2) and RNS, and improved the tolerance of seeds to low-temperature stress. Compared to orthodox seeds such as maize, only transient accumulation of CO and NO was induced and only a moderate increase in GSH was shown in the recalcitrant B. ramiflora seeds. Exogenous CO or NO treatment further increased the GSH accumulation and S-nitrosoglutathione reductase (GSNOR) activity in B. ramiflora seeds under chilling stress. In contrast, suppressing CO or NO generation, removing GSH, or blocking GSNOR activity resulted in increases in ROS and RNS and impaired the germination of CO- or NO-induced seeds under chilling stress. Based on these results, we propose that CO acts as a novel regulator to improve the tolerance of recalcitrant seeds to low temperatures through NO-mediated glutathione homeostasis.     

The encapsulation technology in fruit plants—A review

Encapsulation technology is an exciting and rapidly growing area of biotechnological research. This has drawn tremendous attention in recent years because of its wide use in conservation and delivery of tissue cultured plants of commercial and economic importance. Production of synthetic seeds by encapsulating somatic embryos, shoot buds or any other meristmatic tissue helps in minimizing the cost of micropropagated plantlets for commercialization and final delivery. In most of fruit crops, seed propagation has not been successful because of heterozygosity of seeds, minute seed size, presence of reduced endosperm, low germination rate, and also some are having seedless varieties. Many species have desiccation-sensitive intermediate or recalcitrant seeds and can be stored for only a few weeks or months. Under these circumstances, increasing interest has been shown recently to use encapsulation technology for propagation and conservation. Many fruit plants are studied worldwide for breeding, genetic engineering, propagation, and pharmaceutical purposes. In this context, synthetic seeds would be more applicable in exchange of elite and axenic plant material between laboratories and extension centers due to small bead size and ease in handling. Due to these advantages, interest in using encapsulation technology has continuously been increasing in several fruit plant species. The purpose of this review is to focus upon current information on development of synthetic seeds in several fruit crops.