Genetic load and viability of a future restored northern white rhino population.

As biodiversity loss outpaces recovery, conservationists are increasingly turning to novel tools for preventing extinction, including cloning and in vitro gametogenesis of biobanked cells. However, restoration of populations can be hindered by low genetic diversity and deleterious genetic load. The persistence of the northern white rhino (Ceratotherium simum cottoni) now depends on the cryopreserved cells of 12 individuals. These banked genomes have higher genetic diversity than southern white rhinos (C. s. simum), a sister subspecies that successfully recovered from a severe bottleneck, but the potential impact of genetic load is unknown. We estimated how demographic history has shaped genome-wide genetic load in nine northern and 13 southern white rhinos. The bottleneck left southern white rhinos with more fixed and homozygous deleterious alleles and longer runs of homozygosity, whereas northern white rhinos retained more deleterious alleles masked in heterozygosity. To gauge the impact of genetic load on the fitness of a northern white rhino population restored from biobanked cells, we simulated recovery using fitness of southern white rhinos as a benchmark for a viable population. Unlike traditional restoration, cell-derived founders can be reintroduced in subsequent generations to boost lost genetic diversity and relieve inbreeding. In simulations with repeated reintroduction of founders into a restored population, the fitness cost of genetic load remained lower than that borne by southern white rhinos. Without reintroductions, rapid growth of the restored population (>20-30% per generation) would be needed to maintain comparable fitness. Our results suggest that inbreeding depression from genetic load is not necessarily a barrier to recovery of the northern white rhino and demonstrate how restoration from biobanked cells relieves some constraints of conventional restoration from a limited founder pool. Established conservation methods that protect healthy populations will remain paramount, but emerging technologies hold promise to bolster these tools to combat the extinction crisis.

In Vitro Conservation of Mexican Garlic Varieties by Minimal Growth.

Garlic (Allium sativum L.) is one of the 30 crops that are essential for world food; therefore, its conservation should be considered a priority. There are two main plant conservation strategies: in situ and ex situ conservation. Both strategies are important; nevertheless, ex situ field conservation is affected by biotic and abiotic factors. A complementary strategy to preserve garlic germplasm in the medium term is through in vitro culture by minimal growth. The aim of this study was to evaluate the in vitro conservation of three Mexican garlic varieties by minimal growth. Garlic plants obtained from in vitro garlic bulbs were preserved in six culture media at 25, 18, and 5 °C. A randomized design was used and an analysis of the variance of the survival, contamination, and shoot height of the explants was performed at 30, 60, 90, 180, 270, and 365 days of culture. The results showed that the in vitro conservation of Pebeco, Tacátzcuaro Especial, and Huerteño garlic varieties was optimally obtained for one year at 5 °C in a basal Murashige and Skoog (MS) culture medium with 68.46 g L-1 sucrose and 36.43 g L-1 sorbitol. Thus, the achieved protocol can be adapted to other varieties of garlic for medium-term storage in germplasm banks.

Response of Rowan Berry (Sorbus redliana) Shoot Culture to Slow Growth Storage Conditions.

Slow growth storage can preserve the genetic resources of endangered species such as those of genus Sorbus. Our aim was to study the storability of rowan berry in vitro cultures, their morpho-physiological changes, and regeneration ability after different storage conditions (4 ± 0.5 °C, dark; and 22 ± 2 °C, 16/8 h light/dark). The cold storage lasted for 52 weeks, and observations were made every four weeks. Cultures showed 100% survival under cold storage, and those taken from the storage showed 100% regeneration capacity after the passages. A dormancy period lasting about 20 weeks was observed, followed by intensive shoot growth until the 48th week, which led to the exhaustion of the cultures. The changes could be traced to the reduction of the chlorophyll content and the Fv/Fm value, as well as in the discoloration of the lower leaves and the appearance of necrotic tissues. Long, etiolated shoots (89.3 mm) were obtained at the end of cold storage. Shoot cultures stored in a growth chamber as control (22 ± 2 °C, 16/8 h light/dark) senesced and died after 16 weeks. Explants from stored shoots were subcultured for four weeks. The number and length of newly developed shoots were significantly higher on explants from cold storage compared to those from control cultures if the storage was longer than one week.

Enzymatic Antioxidant System Activation Assures the Viability of Guadua chacoensis (Bambusoideae, Poaceae) Embryogenic Cultures during Cryopreservation.

This study aimed to establish a cryopreservation protocol for G. chacoensis embryogenic cultures (ECs) and to investigate the role of antioxidant enzymes activities during cryopreservation. The growth dynamics of cell suspensions were also investigated, followed by a phytotoxicity test to assess the ECs' ability to tolerate the use of cryoprotective solutions for different incubation times (0, 30, 60, 120, and 240 min). We evaluated the EC redox state in three steps of cryopreservation: after incubation in cryoprotection solution, after thawing, and 60 days after regrowth. Our results showed that the ECs support the use of cryoprotective solution until 120 min, showing phytotoxic effects with 240 min of incubation. This study reports a 100% survival of the cultures and a 10% increase ratio in fresh material for both incubation times tested (60 and 120 min). Increased malonaldehyde content was identified after incubation in the cryoprotective solution. An increase in the activities of catalase and ascorbate peroxidase was also identified in the subsequent steps, suggesting that the activation of antioxidant enzymes is essential for maintaining cell homeostasis during cryopreservation.

Initiative on Avian Primordial Germ Cell Cryobanking in Thailand.

Background: Biobanking the reproductive tissues or cells of animals preserves the genetic and reproductive ability of the species in long-term storage and promotes sharing of reproductive materials. In avian species, the primordial germ cell (PGC) is one of the most promising reproductive cells to be preserved in biobanks, due to self-renewal properties and direct access to the germ line mediated by PGC transfer. Methods: To conserve the genetic resource of local chicken breeds that are of conservation importance, we systematically isolated two types of pregonadal PGCs from chicken embryos-circulating and tissue PGCs. PGCs of individual embryos were separately isolated, cultured, and cryopreserved. Characteristics of cultured PGCs are described and evaluated. Results: The efficiency of PGC isolation from individual embryos was 98.9% (660/667). In most cases, both matching circulating and tissue PGC lines were isolated from the same embryo (68.2%, 450/660), whereas the remaining lines were from a single source, being either tissue (30.6%, 202/660) or circulating (1.2%, 8/660). Efficient PGC isolation and proliferation can be expected in cultures of circulating PGCs (68.7% and 64.3%, respectively) and tissue PGCs (97.8% and 80.7%, respectively). Following cryopreservation, recovered cells sustained PGC identities including expression of chicken vasa homolog and deleted in azoospermia-like proteins and migration ability to recipient embryonic gonads. Culture conditions equally supported proliferation of circulating and tissue PGCs from both sexes. Combining tissue PGC culture in the regimen prevented 30.3% loss of PGC cultures in the case where circulating PGC culture was ineffective. Cultured circulating and tissue PGCs were similar in morphology, but optimal culture characteristics were different. Conclusion: We applied the approach of PGC isolation from blood and tissue origins on a wide scale and demonstrated its efficiency for biobanking chicken PGCs. The workflow can be operated effectively almost year-round in a tropical climate. It was also described in ample and practical details, which are suitable for adoption or optimization in other conditions.

Cryopreservation of Endangered Ornamental Plants and Fruit Crops from Tropical and Subtropical Regions.

Horticultural crops comprise various economic species extending from fruits, nuts, vegetables, spices and condiments, ornamentals, aromatic, and medicinal plants. Ornamental and fruit plants are produced mainly for their nutritional and aesthetic values, respectively. Unfortunately, many tropical and subtropical species are in danger of extinction because of climate change and (a)biotic stresses. It is imperative to preserve the germplasms of these species for the present and future genetic improvement programs. Cryopreservation, i.e., maintenance of tissues at the ultralow temperature of liquid nitrogen, is a promising long-term preservation technique, alternative to seed or in vitro banks, which can be applied for both vegetatively and generatively (through seeds) propagated crops, including those with recalcitrant seeds. It is a technology of choice not only for the preservation of plant biodiversity but also for virus elimination in the proficient administration of large-scale micropropagation. The main advantages of cryopreservation are the lowering of in vitro culture expenditures, needed space, contamination risk, and operator errors. However, tropical species are temperature delicate and one of the foremost challenging issues is preconditioning treatments that stimulate physiological reactions to sufficiently enhance tolerance to dehydration and cryogenic procedures. In recent years, several cryopreservation methods based on encapsulation-vitrification, droplet-vitrification, the use of aluminum cryo-plates, and cryo-mesh have been established. Combined cryo-techniques, gene/DNA conservation, as well as studies on perceiving bio-molecular events and exploring the multistage process from the beginning to end of cryopreservation are receiving more emphasis. The development of cryobiomics delivers a conceptual framework to assess the significance of cell signaling mechanisms on cellular functions, the influence of cryoinjury factors on sample viability, and the implications for genetic stability following cryo-storage. The aim of this mini-review article is to provide a succinct synthesis of the developed cryogenic procedures and their use for the storage and exchange of genetic resources of tropical and subtropical horticultural crops, particularly fruit crops and ornamental plants under the threat of extinction.

Short-Term Storability of Alginate-Encapsulated Persian Violet Microshoots for Germplasm Exchange.

Microshoots have been widely used for micropropagation. It may be necessary to store microshoots for a short period of time, for example in germplasm exchange needing transport to other research groups. Here, we investigated the short-term storability of alginate-encapsulated Persian violet (Exacum affine Balf. f. ex Regel) microshoots at 4 °C and 25 °C. After storage, the encapsulated microshoots were sown on basal Murashige and Skoog medium for germination and viability determination using tetrazolium chloride staining. The results showed that one or five microshoots encapsulated with a single alginate layer could be stored at 4 °C for up to 30 days, while the percentages of germination and viability of the microshoots encapsulated with two layers of alginate were greatly reduced upon storage. This is the first report on the storability of alginate-encapsulated multiple microshoots, which could be a more efficient way to encapsulate microshoots used for short-term cold storage.

Cellular Characterization and Effects of Cryoprotectant Solutions on the Viability of Fibroblasts from Three Brazilian Wild Cats.

Preserving genetic material in cryogenic conditions presents a viable alternative for the protection of species' gene variability. However, there is an enormous need to establish and test cryopreservation protocols that are suitable for each diverse cell type to guarantee technical success in the long run. Considering this, fibroblasts from jaguar (Panthera onca), oncilla (Leopardus tigrinus), and pampas cat (Leopardus colocolo) were subjected to cell characterization and then cryopreservation in different cryoprotectant solutions (2.5%, 10% dimethyl sulfoxide [DMSO] or CryoSOfree™). Further testing was conducted to determine each solution's performance in preserving cell viability. In culture, a growth curve to assess cellular growth potential showed that exponential proliferation lasts for about the first 50 hours of in vitro culturing, declining in pace afterward. L. colocolo and L. tigrinus presented no difference in cell viability while using 2.5% DMSO protocols. P. onca cells did not present difference on viability for both concentrations of DMSO. Protocols using CryoSOfree resulted in a decreased viability of P. onca fibroblasts. Morphological differences between fibroblasts among the species were noted under bright field microscopy and scanning electron microscopy. L. colocolo and P. onca cells are fusiform, and L. tigrinus are spherical. All cells presented cytoplasmic projections. Transmission electron microscopy revealed vacuoles and secretion granules, indicating intense cell activity after thawing. Differences found in the efficiency of cryopreservation protocols according to the type of cryoprotectant indicate that species react differently to freezing and thawing processes. This research evaluates key aspects of in vitro protocols for cryopreservation of wild animals, which need to be optimized to guarantee successful cell culturing. More suitable protocols lead to increased efficiency in establishing fibroblast cryobanks and also facilitating the use of wild cats' cells in cloning techniques, contributing directly to preserving wild fauna.

Challenges and Prospects for the Conservation of Crop Genetic Resources in Field Genebanks, in In Vitro Collections and/or in Liquid Nitrogen.

The conservation of crop genetic resources, including their wild relatives, is of utmost importance for the future of mankind. Most crops produce orthodox seeds and can, therefore, be stored in seed genebanks. However, this is not an option for crops and species that produce recalcitrant (non-storable) seeds such as cacao, coffee and avocado, for crops that do not produce seeds at all; therefore, they are inevitably vegetatively propagated such as bananas, or crops that are predominantly clonally propagated as their seeds are not true to type, such as potato, cassava and many fruit trees. Field, in vitro and cryopreserved collections provide an alternative in such cases. In this paper, an overview is given on how to manage and setup a field, in vitro and cryopreserved collections, as well as advantages and associated problems taking into account the practical, financial and safety issues in the long-term. In addition, the need for identification of unique accessions and elimination of duplicates is discussed. The different conservation methods are illustrated with practical examples and experiences from national and international genebanks. Finally, the importance of establishing safe and long-term conservation methods and associated backup possibilities is highlighted in the frame of the global COVID-19 pandemic.

Conserving and Sharing Taro Genetic Resources for the Benefit of Global Taro Cultivation: A Core Contribution of the Centre for Pacific Crops and Trees.

This review article gives an account of the origin, domestication, and dispersal of taro, a staple food crop in many countries in the humid tropics and subtropics. Genetic diversity studies indicated that distinct gene pools exist in all the regions where taro may be naturally distributed-the Indian subcontinent, China, Southeast Asia, and in Oceania. The Asian gene pool presented the highest genetic diversity. Diploid taro is prevalent in the Pacific Islands, while both diploids and triploids are found in mainland Asia. Triploids are thought to provide better adaptability and enhanced hardiness to higher altitudes and latitudes where sexual reproduction is not viable. The Centre for Pacific Crops and Trees (CePaCT) conserves in vitro close to 70% of the taro genetic resources held ex situ and is therefore considered the world center for taro genetic resources. Phytophthora colocasiae or taro leaf blight (TLB) is the most severe disease of taro' causing 25%-50% yield losses and postharvest decay of corms. The CePaCT genebank supported the participatory TLB breeding program in Samoa through the provision of diverse taro germplasm from the Asian gene pool. However, CePaCT not only serves taro producers in the Pacific but also shares new allelic diversity of taro globally. More recent distributions of taro genetic diversity to West and Central Africa were in response to an outbreak and spread of TLB in West Africa. Global dissemination of taro genetic diversity is assisting producer countries in the process of adaptation to emerging biotic and abiotic stresses, exacerbated by climate change.

Cryopreservation of banana's cv Grand Naine in vitro rhizomes

ABSTRACT The preservation of banana genetic material is usually performed through seedlings. However, most banana cultivars do not produce seed and are propagated vegetatively. Therefore, cryopreservation is a feasible technique that allows the preservation of banana genotypes indefinitely. For the success of cryopreservation protocols, the selection of cryoprotectants and pre-freezing techniques are important factor. Therefore, the objective of this study was to verify the effects of different cryoprotectants with and without 1% phloroglucinol and pre-cooling periods on the development of a protocol for cryopreservation of in vitro rhizomes ofMusa accuminata(AAA) cv Grand Naine banana. The addition of 1% phloroglucinol to the cryoprotective solutions, such as PVS2 enhanced recovery of cryopreserved banana rhizomes. In addition, pre-cooling of explants in ice for 3 hours in PVS2 + 1% of phloroglucinol allowed efficient cryopreservation of banana rhizomes, followed by successful recovery and regeneration of in vitro shoots of banana cv Grand Naine.

Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production.

Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as "Indian Ginseng", is a medicinally and industrially important plant of the Indian subcontinent and other warmer parts of the world. The plant has multi-use medicinal potential and has been listed among 36 important cultivated medicinal plants of India that are in high demand for trade due to its pharmaceutical uses. The medicinal importance of this plant is mainly due to the presence of different types of steroidal lactones- withanolides in the roots and leaves. Owing to low seed viability and poor germination, the conventional propagation of W. somnifera falls short to cater its commercial demands particularly for secondary metabolite production. Therefore, there is a great need to develop different biotechnological approaches through tissue and organ culture for seasonal independent production of plants in large scale which will provide sufficient raw materials of uniform quality for pharmaceutical purposes. During past years, a number of in vitro plant regeneration protocols via organogenesis and somatic embryogenesis and in vitro conservation through synthetic seed based encapsulation technology have been developed for W. somnifera. Several attempts have also been made to standardize the protocol of secondary metabolite production via tissue/organ cultures, cell suspension cultures, and Agrobacterium rhizogenes-mediated transformed hairy root cultures. Employment of plant tissue culture based techniques would provide means for rapid propagation and conservation of this plant species and also provide scope for enhanced production of different bioactive secondary metabolites. The present review provides a comprehensive report on research activities conducted in the area of tissue culture and secondary metabolite production in W. somnifera during the past years. It also discusses the unexplored areas which might be taken into consideration for future research so that the medicinal properties and the secondary metabolites produced by this plant can be exploited further for the benefit of human health in a sustainable way.

Rapid responses of plants to temperature changes.

Temperature is one of the main environmental factors that affect plant metabolism. Considering that plants are sessile, their survival depends on the efficient activation of resistance responses to thermal stress. In this comprehensive review, we discuss recent work on rapid biochemical and physiological adjustments, herein referred to as those occurring during the first few hours or a few days after the beginning of the change in the ambient temperature. The short-term metabolic modulation after plant exposure to heat and cold, including chilling and freezing, is discussed. Effects on photosynthesis, cell membranes, antioxidant system, production of heat shock proteins and nitric oxide, as well as an overview of signaling events to heat or cold stress are presented. In addition, we also discuss the acclimation process that occurs when the plant acquires resistance to an increase or decrease in temperature, adjusting its homeostasis and steady-state physiology to the new temperatures. Finally, we present studies with tropical plants that aim at elucidating the effects of temperature and the identification of the resilience levels of these plants to the expected climate changes, and which seek the development of techniques for germplasm conservation of endangered species.

Anatomía comparada de plantas de Dioscorea alata L. clon Caraqueño cultivadas en tres ambientes de crecimiento in vitro / Compared anatomy of Dioscorea alata L. clone Caraqueño plants cultivated in three in vitro growth environments

La conservación in vitro de Dioscorea alata L. clon Caraqueño es fundamental para garantizar la propagación y distribución de material de plantación sano a los productores, y disponer de un banco in vitro de un clon de gran valor agronómico y comercial en la región oriental de Cuba. Con el fin de evaluar las modificaciones anatómicas que se producen en plantas de ñame en tres condiciones de cultivo in vitro: plantas conservadas por métodos de mínimo crecimiento, plantas regeneradas y plantas en fase de multiplicación en el medio MS 75 %, se realizó un análisis de la anatomía foliar y caulinar a partir de cortes transversales de la lámina foliar y del tallo, y cortes longitudinales y transversales de microtubérculos formados durante el proceso de conservación. Las hojas de las plantas conservadas mostraron menor espesor del mesófilo y la epidermis y el área de los haces conductores del tallo también fue menor, debido al proceso de stress durante la conservación in vitro. Sin embargo, durante la recuperación del material conservado a través de la regeneración y la multiplicación in vitro se restablecieron de manera normal estos parámetros. También se evidenció que los microtubérculos formados en la conservación in vitro, poseen parénquima amilífero con abundantes gránulos de almidón, capa delgada de parénquima cortical, y haces conductores poco desarrollados, todo lo cual indica la presencia de actividad meristemática.

The in vitro conservation of Dioscorea alata L. clone Caraqueño is fundamental to guarantee the propagation and distribution of healthy plantation material to the farmers and the establishment of one in vitro bank of this clone of great agronomic and commercial value in the Oriental Region of Cuba. With the purpose of evaluating the anatomical modifications that take place in yam plants under three in vitro culture conditions: conserved plants by slow growth, regenerated plants and in plants multiplication phase in MS 75% medium, was carried out an analysis of the foliar and caulinar anatomy from transversal cuts of the foliar sheet and of the stem, and longitudinal and transversal cuts of microtubers formed during the conservation process. Smaller thickness of the mesophyll and of the epidermis in the leaves of the conserved plants were showed and the conductive sheaves area of the stem were also smaller, due to the stress process during the in vitro conservation. However during the recovery of the conserved material through the regeneration and the in vitro multiplication were reestablished to their normal state these parameters. It was also evidenced that the microtubers formed in the in vitro conservation, have reserve parenchyma with abundant starch granules, thin cortical parenchyma and conductive sheaves little developed were determined. All this characteristics indicated the presence of meristematic activity.

Protocols for Biotechnological Interventions in Improvement of Vanilla (Vanilla planifolia Andrews.).

Vanilla (Vanilla planifolia Andrews (syn. V. fragrans Salisb.), a native of Central America, is the primary source of natural vanillin and plays a major role in the global economy. The gene pool of vanilla is threatened by deforestation and overcollection that has resulted in disappearance of natural habitats and wild species. Continuous vegetative propagation and lack of natural seed set and sufficient variations in the gene pool hamper crop improvement programs. In vitro techniques, one of the key tools of plant biotechnology, can be employed for overcoming specific problems, viz. production of disease-free clones, inducing somaclonal variations, developing hybrids, gene pool conservation, incorporating desired traits by distant hybridization, genetic engineering, etc. However, realization of these objectives necessitates standardization of protocols. This chapter describes the various protocols optimized for crop improvement in Vanilla species.

In Vitro Propagation and Conservation of Bacopa monnieri L.

Bacopa monnieri L. (common name brahmi) is a traditional and renowned Indian medicinal plant with high commercial value for its memory revitalizer potential. Demand for this herb has further escalated due to popularization of various brahmi-based drugs coupled with reported anticancer property. Insufficient seed availability and problems associated with seed propagation including short seed viability are the major constraints of seed conservation in the gene banks. In vitro clonal propagation, a prerequisite for in vitro conservation by enhanced axillary branching was standardized. We have developed a simple, single step protocol for in vitro establishment, propagation and medium-term conservation of B. monnieri. Single node explants, cultured on Murashige and Skoog's medium supplemented with BA (0.2 mg/L), exhibited shoot proliferation without callus formation. Rooting was achieved on the same medium. The in vitro raised plants were successfully transferred to soil with ~80 % survival. On the same medium, shoots could also be conserved for 12 months with high survival and genetic stability was maintained as revealed by molecular markers. The protocol optimized in the present study has been applied for culture establishment, shoot multiplication and medium-term conservation of several Bacopa germplasm, procured from different agro-ecological regions of India.

Protocols for Improvement of Black Pepper (Piper nigrum L.) Utilizing Biotechnological Tools.

Black pepper, Piper nigrum L., the "King of spices" is the most widely used spice growing in the South-Western region of India. The humid tropical evergreen forest bordering the Malabar Coast (Western Ghats is one of the hot spot areas of plant bio-diversity on earth) is its center of origin and diversity. However, the crop faces constraints like rampant fungal and viral diseases, lack of disease free planting material, hence biotechnological tools can be utilized to address these problems and strides have been made successfully. The standardization of micropropagation, somatic embryogenesis, in vitro conservation, protoplast isolation, and genetic transformation protocols are described here. The protocols could be utilized to achieve similar goals in the related species of Piper too.

Protocols for In Vitro Propagation, Conservation, Synthetic Seed Production, Microrhizome Production, and Molecular Profiling in Turmeric (Curcuma longa L.).

Turmeric is a rhizomatous herbaceous perennial but cultivated as annual, belonging to the family Zingiberaceae. It is a native of India and South East Asia. The tuberous rhizomes or underground stems of turmeric are used from antiquity as condiments, a dye and as an aromatic stimulant in several medicines. Turmeric is an important crop in India and it is used as a spice, food preservative, coloring agent, cosmetic as well as for its medicinal properties. Propagation is done vegetatively with rhizome bits as seed materials. It is plagued by rhizome rot diseases most of which are mainly spread through infected seed rhizomes. Micropropagation will help in production of disease-free seed. Sexual reproduction is rare in turmeric, making recombinant breeding very difficult. In vitro technology can thus become the preferred choice and it can be utilized for multiplication, conservation of genetic resources, generating variability, gene transfer, molecular tagging, and their utility in crop improvement.

Protocols for In Vitro Propagation, Conservation, Synthetic Seed Production, Embryo Rescue, Microrhizome Production, Molecular Profiling, and Genetic Transformation in Ginger (Zingiber officinale Roscoe.).

Ginger is a rhizomatous plant that belongs to the family Zingiberaceae. It is a herbaceous perennial but cultivated as annual, with crop duration of 7-10 months. Ginger is native to India and Tropical South Asia. The tuberous rhizomes or underground stems of ginger are used as condiment, an aromatic stimulant, and food preservative as well as in traditional medicine. Ginger is propagated vegetatively with rhizome bits as seed material. Cultivation of ginger is plagued by rhizome rot diseases, most of which are mainly spread through infected seed rhizomes. Micropropagation will help in production of disease-free planting material. Sexual reproduction is absent in ginger, making recombinant breeding very impossible. In vitro technology can thus become the preferred choice as it can be utilized for multiplication, conservation of genetic resources, generating variability, gene transfer, molecular tagging, and their utility in crop improvement of these crops.

Micropropagation and in vitro conservation of the rare and threatened plants Ramonda serbica and Ramonda nathaliae.

Ramonda serbica and Ramonda nathaliae are rare and endemo relict plant species from Balkan Peninsula. An efficient micro propagation and in vitro conservation method via direct and indirect organogenesis from seed and leaf explants, respectively, was established in this study. The seed of both Ramonda species were collected from different populations in Kosovo, and were germinated in nutrient media JG-B without any phytohormone. The highest number of shoots and multiplication rate was observed on JG-B medium supplemented with BAP and IAA (0.5 mg l(-1) each), whereas the highest number of leaves per plantlets was found on WPM and RA medium supplemented with BAP and IAA (0.1 mg l(-1) each). During this stage of micro propagation some significant differences were observed in plantlets from different populations. The indirect organogenesis from parts of leaves of natural plants was not successful due to unavailability of established protocol for disinfections of the plant material. On other hand, parts of leaves from micro propagated plantlets, cultured on MS medium supplemented with different ratio of BAP and NAA, resulted in the highest efficiency for shoot regeneration. In vitro conservation of micro propagated plants at the lower temperature (4 °C) had a significantly positive effect for storage of more than 12 months.