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Fruit trees provide essential nutrients to humans by contributing to major agricultural outputs and economic growth globally. However, major constraints to sustainable agricultural productivity are the uncontrolled proliferation of the population, and biotic and abiotic stresses. Tree mutation breeding has been substantially improved using different physical and chemical mutagens. Nonetheless, tree plant breeding has certain crucial bottlenecks including a long life cycle, ploidy level, occurrence of sequence polymorphisms, nature of parthenocarpic fruit development and linkage. Genetic engineering of trees has focused on boosting quality traits such as productivity, wood quality, and resistance to biotic and abiotic stresses. Recent technological advances in genome editing provide a unique opportunity for the genetic improvement of woody plants. This review examines application of the CRISPR-Cas system to reduce disease susceptibility, alter plant architecture, enhance fruit quality, and improve yields. Examples are discussed of the contemporary CRISPR-Cas system to engineer easily scorable PDS genes, modify lignin, and to alter the flowering onset, fertility, tree architecture and certain biotic stresses.

Date palm (Phoenix dactylifera L.) is an economically important crop in the oases agroecological zones. In vitro mutagenesis has been an effective strategy for genetic improvements in several traits of crop plants. However, studies related to the genetic improvement of this fruit tree are very limited. Several conventional approaches including physical and chemical mutagens, insertional and somaclonal mutations have been practiced creating the desired traits. However, contemporary site-directed mutation approaches, like TALENS, ZNFs and CRISPR-Cas, have not yet been put into practice for the date palm. The prospects and applications of currently accessible mutagenesis methods for date palm genetic improvement are discussed in this chapter. New breeding tools (NBTs) for targeted mutagenesis through CRISPR-Cas-based genome editing (GE) and its base editing (BE) versions can be very effective to engineer date palm genomes. However, with a large and complex genome, heterozygosity and outcrossing, somaclonal variation during in vitro regeneration, the presence of single-nucleotide polymorphism (SNP) and ultimate genetic instability caused by these SNPs pose challenges. Such challenges could be addressed effectively by the execution of site-specific CRISPR-Cas versions, like BEs, coupled with high-throughput screening techniques. Finally, the hierarchy of targeted mutagenesis over random mutagenesis is addressed as a potential approach for futuristic studies of date palm genetic improvement.

In arid and semiarid areas, date palm is an essential fruit crop. Date is considered an ideal food with numerous nutritional and medicinal properties. Date is rich in sugar, protein, dietary fiber, minerals, and some vitamins. Its pulp, pit, pollen, and spathe extracts were used to prevent a wide range of chronic diseases. It contains a broad range of secondary metabolites, including anthocyanins, carotenoids, flavonoids, phenolic acid, procyanidins, and sterols. Date bioactive compounds are antioxidants, antimicrobial, anticancer, anti-inflammatory, and hepatoprotective. Biosynthesis of these compounds is influenced by several factors such as sunlight, irrigation, cultivar, tissue type, pollination, thinning, and maturity stage. This chapter discusses the medicinal and nutritional value of dates with an emphasis on secondary metabolites and various factors influencing their biosynthesis, pharmaceutical benefits, and potential use in functional food. Recent advances in metabolomics are also discussed.

Plants generally have the highest regenerative ability because they show a high degree of developmental plasticity. Although the basic principles of plant regeneration date back many years, understanding the cellular, molecular, and physiological mechanisms based on these principles is currently in progress. In addition to the significant effects of some factors such as medium components, phytohormones, explant type, and light on the regeneration ability of an explant, recent reports evidence the involvement of molecular signals in organogenesis and embryogenesis responses to explant wounding, induced plant cell death, and phytohormones interaction. However, some cellular behaviors such as the occurrence of somaclonal variations and abnormalities during the in vitro plant regeneration process may be associated with adverse effects on the efficacy of plant regeneration. A review of past studies suggests that, in some cases, regeneration in plants involves the reprogramming of distinct somatic cells, while in others, it is induced by the activation of relatively undifferentiated cells in somatic tissues. However, this review covers the most important factors involved in the process of plant regeneration and discusses the mechanisms by which plants monitor this process.

Banana and plantains are one of the important fruit crops grown extensively in the tropical and subtropical regions of the world. The world production of banana is 145 million tons of which only a few million tons is exported, which means that most production is primarily for local consumption. The banana cultivars are derived from two diploid wild species, Musa acuminata (AA genome) and Musa balbisiana (BB genome). Majority of the edible banana cultivars are propagated vegetatively, and hence, the improvement of banana through conventional breeding methods is difficult. Attempts have been made to improve banana by inducing genetic variability by using both physical and chemical mutagens and exploiting the somaclonal variation a few varieties have been released for cultivation. Transgenic approach has also been used to incorporate the desirable traits into banana. Recent advances in genomics and the availability of genome sequence of both Musa acuminata and Musa balbisiana helps in the improvement of this fruit crop. Also the recent reports of genome editing through CRISPR-CAS9 will aid in speeding up the banana improvement programmes in the near future. This review summarizes the various advances made in inducing genetic diversity in banana.

The rapid loss of genetic diversity has become a major concern worldwide for the genetic improvement of crops for sustainable agriculture especially under the climate change. Moreover, events like rapid human population growth, industrialization, deforestation, and natural calamities, are some of the major factors in this loss. The conservation, distribution, and utilization of natural and induced genetic diversity have become essential by the establishment of gene/germplasm bank both at the national and international levels. In vitro conservation techniques such as cryopreservation and low temperature storage are being routinely used for genetic resources conservation of a wide range of crops including seed propagated and vegetatively propagated crops. Cold-storage is done at 4-5°C and has a major disadvantage of frequent subcultures of in vitro cultures; may run into a risk of contamination. This method is routinely used for seed storage by national and international gene banks, maintained by CGIAR institutes, e.g., International Rice Research Institute (IRRI). Cryopreservation of genetic material is done in liquid nitrogen at -196°C on the long-term basis without going through frequent subcultures. In vitro cultures are suitable for cryo-storage, that includes somatic embryos, cell suspension, callus, and should be able to regenerate plants maintaining their genetic fidelity. In date palm, in vitro conservation work done is very limited. Nowadays, the date palm tissue culture system is well established via somatic embryogenesis and organogenesis, which is of course genotypic dependent on frequency of plant regeneration rate. Most commonly somatic embryogenic cultures have been used for cryo-storage of date palm genetic material. INTRODUCTION Date palm (Phoenix dactylifera) is one of the most ancient cultivated plants since over 4000 years. The continuous selection for desirable traits by man has resulted in narrowing the genetic diversity, and has left hardly any scope for developing new cultivars with useful traits. Very few plant species have been so closely connected with the survival and well being of humans living in hot and arid environments. Also, the date palm tree has a great socio-economic importance and nutritional value in the Middle East and north Africa. In addition, date palm trees withstand adverse environmental changes such as drought, rains, flood, temperature fluctuations, and also help in preventing desertification. These drastic climatic changes could have adverse consequences by losing the genetic diversity. Date fruits are a very important source of human nutrition as well as an export item for date palm growing countries. Most importantly the plant contributes in the creation of a ‘microclimate’ within the fragile oasis ecosystem and allows development of agriculture in drought and saline conditions; multiple cropping system such as grapes, tomato and so on. The genetic diversity of date palm represents different local cultivars grown under varied climatic conditions. The most popular Sudanese date palm cultivars described as soft and dry types, showed detectable genetic variation in fruit morphology (Elshibi, 2009; Jain, 2011). Moreover no conventional methods e.g., seed storage were used to conserve genetic diversity due to high nature of heterozygosis for a mohan.jain@helsinki.fi Proc. 4 Int. Date Palm Conference Eds.: A. Zaid and G.A. Alhadrami Acta Hort. 882, ISHS 2010 786 developing date palm breeding programs. With the development of in vitro culture techniques organogenesis, somatic embryogenesis, embryo rescue-plant regeneration has already been accomplished, which is highly genotypic dependent; has made possible shortand long-term storage of in vitro cultures, large-scale multiplication of plants of elite genotypes (Jain, 2011). CONSERVATION OF GENETIC DIVERSITY Plant genetic diversity conservation has become an important issue among researchers worldwide for present and future agriculture. The rapid loss of genetic resources is mainly due to rapid industrialization, fire, deforestation, and environmental pollution. Moreover, lack of availability or non-existence of desirable genotypes hampers plant breeders for developing new cultivars. Seed source is commonly used to conserve plant genetic resources and stored at low temperature. It is quite cost effective, safer, and easy to handle. The second approach is in vitro conservation of in vitro cultures in cold storage and cryo-storage (Bekheet et al., 2001, 2005, 2007). The success of this approach is very much dependent on plant regeneration and the maintenance of genetic fidelity of the stored genotypes. Nowadays international germplasm exchange has become more cumbersome and sometimes it is rather difficult to obtain elite genotypes. Many countries do not share the germplasm because of patent and ownership problems, and have established national germplasm conservation or gene bank facilities. In India, the National Bureau of Plant Genetic Resources (NBGR) was established in Delhi to conserve local and imported genetic material for proper utilization in developing new cultivars. Countries like Costa Rica, South Korea, Thailand and others have established germplasm conservation facilities. Gene banks of CGIAR institutes worldwide are an important source of exchange of germplasm (Jain, 2011). In the European Union, there are several projects being carried out on germplasm identification, collection, conservation, and utilization of various crops, e.g., Allium spp., and have established websites of available genetic resources. CONSERVATION OF INDUCED GENETIC DIVERSITY The natural genetic diversity is gradually eroded and consequently loss of valuable genetic resources that hinders genetic improvement of crops including date palm. Moreover the rate of spontaneous mutations is extremely slow and makes the availability of wide genetic diversity harder for plant breeders. Therefore, induced mutations hasten the rate of genetic diversity in a short period and are readily available to plant breeders for developing new cultivars. Mutations are induced by physical (e.g., gamma radiation) and chemical mutagen (e.g., sodium azide) treatment in seed and vegetative propagated crops including the date palm (Jain, 2010a,b; Jain et al., 2010). The FAO/IAEA Mutant Variety Database (MVD) collects information on plant mutant varieties (cultivars) released officially or commercially worldwide (http://www.iaea.org). A mutant variety is a new plant variety that is bred through: 1. direct use of a mutant line that is developed through physical and chemical mutagenesis, or somaclonal variation; 2. indirect use of a mutant line/lines, which is/are used as a parental variety/varieties in cross breeding (cross between mutant lines or with a commercial cultivar/cultivars); 3. the use of a mutant gene allele (trait) Calrose 76 sd1 allele (semi-dwarf 1 trait) in rice; 4. use of wild species’ genes translocated into plant genomes through irradiation facilitated translocations, e.g., genes of wheat wild relative species. In the FAO/IAEA mutant variety database, date palm mutants are yet to be available. However, mutation work on date palm has produced putative mutants showing tolerance to Bayoud disease (Jain, 2007).

Prospects of Biotechnology for Tea Improvement

Induction of mutations is an effective approach for enhancing natural genetic resources. FAO/IAEA Coordinated Research Projects (CRP) have been established on tropical and subtropical fruits as well as on banana, and the Technical Co-operation (TC) sub-Regional Project on date palm. The objectives are to improve these fruit crops by induced mutations. These programs have four major components: in vitro culture, radiation induction of mutations, mutation screening, and characterization of mutants with molecular markers. In most of the important fruit crops, tissue culture is well established for plant regeneration either via somatic embryogenesis or organogenesis. Since somatic embryo cultures often originate from a single cell, it is an ideal system for induction of mutations as it helps in preventing chimerism. The rate of somatic embryo germination is very poor, which has become a major hurdle for large-scale plant multiplication of desirable induced mutants. The major focus will be on the development of mutant lines of fruits with improved tolerance against abiotic and biotic stresses, seedlessness e.g. in guava and citrus.

Agricultural sustainability and food security are major challenges facing continued population growth. Integration of existing and new technologies for the induction and exploitation of genetic diversity towards developing healthier, nutritious and productive crops is the need of the hour. Mutagenesis is a proven technology for the development of improved or novel varieties with desirable traits. Several mutant genes have been successfully explored, either directly or indirectly, to complement crop productivity. The advent of genomics approaches and plant genome sequencing has benefitted mutation discovery and mutant characterization. Plant mutant repositories are being established to serve as platforms for basic and applied research in crop improvement. This review briefly outlines the impact and molecular/genomic characterization of induced mutations in crop improvement.

Tissue culture generates a wide range of genetic variation in plant species which can be incorporated in plant breeding programmes. By in vitro selection, mutants with useful agronomic traits, e.g. salt or drought tolerance or disease resistance, can be isolated in a short duration. The successful use of somaclonal variation is very much dependent on its genetic stability in the subsequent generations for which molecular markers such as RAPDs, AFLPs, SSRs and others can be helpful. The potential of somaclonal variation has yet to be fully exploited by breeders, even though a few cultivars have been developed in crops such as Brassica juncea, rice and others.

International audience; While ornamental plants are produced mainly for their aesthetic value, the propagation and improvement of quality attributes such as leaf types, flower colour and fragrance, longevity and form, plant shape and architecture, and the creation of novel variation are important economic goals for the expanding ornamental industry. In Protocols for In Vitro Propagation of Ornamental Plants, leading researchers in the field compile step-wise protocols for rapid plant multiplication and in vitro storage of major commercially viable ornamental plants. Divided into two sections, Section A contains chapters mainly on micropropagation of cut and pot flowers with detailed protocols involving in vitro culture-explants, medium preparation, detailed medium table, shoot initiation and proliferation, root induction, in vitro plant hardening, and field transfer, and Section B delves into reviews on topics such as in vitro production of sweet peas, the status of transgenics in ornamental plants, in vitro conservation, the status of floriculture in Europe, azalea phylogeny, and thin cell layers. As part of the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials, step-by-step laboratory protocols, and notes on troubleshooting and avoiding known pitfalls.Comprehensive and clear, Protocols for In Vitro Propagation of Ornamental Plants presents key techniques that will be of great use to floriculturists, researchers, commercial companies, biotechnologists, and students dealing with ornamental plants.

Strawberry shoot meristem cultures have been used for producing virus-free strawberry plants. Plantlet regneration from leaf mesophyll protoplasts, and Agrobacterium tumefaciens mediated genetic transformation, mark the beginning of strawberry improvement with biotechnologies. The use of biotechnological tools has a greater potential in producing strawberry plants with traits such as early maturation, frost and disease resistance, mite and nematode resistance, high yield, better quality, suitability for mechanical harvesting, shipping ability, ellgaic acid content, aroma compounds and cost-effective micropropagation. Gene identification and mapping with RFLPs or RAPDs would be desirable. Transgenic strawberries should be tested for their quality before releasing to the consumers.

Advances in gene transfer technologies have enabled the production of both monocot and dicot transgenic plants. With the biolistic method, genes can be transferred in recalcitrant crop plants and forest trees, independent of their genotype. Inexpensive methods for both stable and transient gene transfers - ultrasonication, direct DNA insertion during imbibition using somatic embryos, and silicon carbide fibres - have been developed. The frequency of Agrobacterium-mediated transformation rates of cloned genes can be enhanced in plant cells. The analysis of molecular markers (RFLPs, RAPDs, DNA fingerprints) can accomplish the characterization, gene mapping and identification and certification and patent protection of cultivars. With PCR, selective amplification of a specific DNA segment from a small amount of an organism’s total DNA can be used toidentify transgenic cultivars. The expression of a target gene can be inhibited with antisense RNA. So far, a limited number of genes have been identified and cloned with genetic engineering. With specific gene transfers, many goals such as biological control of insect pests and fungi, male sterility, virus resistance, improving seed protein, and production of transgenic plants as “bioreactors” can be accomplished. T-DNA mutagenesis may lead to learning more about the genetic control of plant development and morphogenesis, and isolation of useful mutants. Before genetic engineering becomes a reliable tool of plant breeding, more attention is needed to explore: (a) new plant genetic resources in order toidentify and clone new genes, (b) fate of selective and scorable marker genes, and (c) field evaluation of transgenes in transgenic plants., Geeninsiirtoteknologiassa tapahtunut edistys on tehnyt mahdolliseksi yksi- ja kaksisirkkaisten siirtogeenisten kasvien tuotannon. Biolistisella menetelmällä voidaan siirtää geenejä vaikeasti käsiteltäviin viljelykasveihin ja metsäpuihin riippumatta niiden genotyypistä. Pysyvää ja lyhytaikaista geenien siirtoa varten on kehitetty halvempia menetelmiä: ultraäänimetodi, suora DNA-siirto liotusvaiheessa käyttäen kuivia somaattisia alkioita ja silikoni karbiidikuidut. Agrobacterium -välitteistäkloonattujen geenien siirtoa kasvisoluihin voidaan tehostaa. Molekyylimerkkien - RFLP’t, RAPD’t ja DNA-sormenjälki - analysointi voi täydentää viljelykasvien kuvailua, geenikartoitusta ja tunnistamista, takuutodistuksen antoa sekä patenttien suojaamista. PCR:n avulla voidaan valikoidusti monistaa spesifinen DNA-segmentti ottamalla vain pieni määrä organismin koko DNA:sta ja tunnistaa siirtogeeninen viljelykasvi. Kohteena olevan geenin toiminta voidaan estää antisense RNA´lIa. Toistaiseksi geeniteknologian avulla on tunnistettu ja kloonattu rajoitettu määrä geenejä. Spesifillä geeninsiirrolla päästään moniin päämääriin: tuhohyönteisten ja sienien biologiseen torjuntaan, koiras-steriiliyteen, viruskestävyyteen, siemenproteiinien parantamiseen ja siirtogeenisten kasvien käyttöön bioreaktoreina. T-DNA mutageneesin avulla voidaan saada tietoa kasvin kehityksen ja morfogeneesin geneettisestä säätelystä sekä eristää hyödyllisiä mutantteja. Ennen kuin geeniteknologia voi olla luotettava keino jalostaa kasveja, olisi kiinnitettävä enemmän huomiota kasvien geneettisten varojen selvittämiseen, jotta voitaisiin löytää ja kloonata uusia geenejä. Samoin olisi selvitettävä selektiivisten ja raportoivien merkkigeenien vaikutuksia kasveissa ja tutkittava kenttäoloissa siirtogeenejä ja siirtogeenisiä kasveja.

Preface. 1. Molecular markers: principles and methodology P.K. Gupta, et al. 2. Microsatellites and molecular breeding: exploitation of microsatellite variability for the analysis of a monotonous genome P. Winter, et al. 3. Development and application of molecular markers in conifers R. Schubert, et al. 4. Molecular markers and heterosis P.K. Ranjekar, et al. 5. Molecular markers and abiotic stresses I. Winicov. 6. Molecular markers for flowering time genes in crop species D.A. Laurie, S. Griffiths. 7. Molecular markers for the genetic analysis of apomixis M.J. Asins, et al. 8. Use of molecular markers for fruit crop improvement A.R. McCaskill, J.J. Giovannoni. 9. In situ hybridization in plants- methods and application, J. Maluszynska. 10. Molecular tools for improving coffee (Coffea arabica L.) resistance to parasites D. Fernandez, P. Lashermes. 11. Construction and use of genetic maps in cereals M. Motto, P.A. Marsan. 12. Gene expression under environmental stresses-molecular marker analysis M. Brosche, et al. 13. Random insertional mutagenesis in Arabidopsis T. Ito, K. Shinozaki. 14. Engineering the cloroplast genome to confer stress tolerance and production of pharmaceutical proteins H. Daniell. 15. Identification of strawberry flavour related genes by the use of DNA microarrays A. Aharoni, A.P. O'Connell. 16. Gene targeting in plants S. Kumar, M. Fladung. 17. Plant DNA methylation and gene expression M. Bellucci, et al. 18. QTL mapping in crop plants S.B. Andersen, A.M. Torp. 19. QTLs for root growth and drought resistance in rice A. Price. 20. QTL mapping for forage traits Th. Lubberstedt. 21. QTLs in bread making quality a review G. Charmet, C. Groos.

Somatic embryogenesis has the vast potential to produce plants in their millions, and has now become a routine protocol for many trees. However, the use of SE in a wide range of woody plants is yet to be utilized due to several limitations facing the process, including poor germination of somatic embryos, genotypic influences, a limited number of explants inducing SE, and somaclonal variation. These limitations have hindered the commercialization of SE in a wide range of forest trees. Further research is required at both the biochemical and molecular levels to understand the mechanism of SE, enabling the induction of SE in other recalcitrant woody species. A sharp focus is needed to increase the rate of ESM establishment, improving yield from established ESM, and increasing the germination rate of somatic embryos for commercialization, without which this technology will have very limited scope (Timmis 1998). Automation of somatic-seed production is the ultimate goal of commercial seed industries. The combination of high technology with tissue culture, including computer-aided image analysis, robotics, bioreactors (including temporary immersion systems) somatic-embryo encapsulation, development of appropriate somatic-embryo coating material, etc., require further investment for producing millions of somatic seeds in a short time and to cut down the cost of seed production. The genetic fidelity of somatic seedlings is important for all of this, which can be tested with molecular markers, such as AFLPs or microsatallites. Although several molecular markers are available, a reliable molecular diagnostic kit is still needed for the early detection of genetic variability during plant development.

Section A -- Slash Pine (Pinus elliottii Engelm.) -- Somatic Embryogenesis and Genetic Transformation in Pinus radiata -- Douglas - Fir (Pseudotsuga menziesii) -- Omorika Spruce (Picea omorika) -- Somatic Embryogenesis in Picea glauca -- Protocol of Somatic Embryogenesis: Black Spruce (Picea mariana (Mill.) B.S.P.) -- Sitka Spruce (Picea sitchensis) -- Protocol of Somatic Embryogenesis of Pinus nigra Arn. -- Loblolly Pine (Pinus taeda) -- Somatic Embryogenesis in Maritime Pine (Pinus pinaster Ait.) -- Somatic Embryogenesis in Pinus patula -- Somatic Embryogenesis in Norway Spruce -- Section B -- Cashew (Anacardium occidentale L.) -- Somatic Embryogenesis Protocol: Coffee (Coffea arabica L. and C. canephora P.) -- Protocols for Somatic Embryogenesis and Plantlet Formation from Three Explants in Tea (Camellia sinensis (l.) o. kuntze) -- Protocol of Somatic Embryogenesis from Citrus spp. Anther Culture -- Integrated System for Propagation of Theobroma cacao L. -- Mango (Mangifera indica L.) -- Somatic Embryogenesis Injackfruit (Artocarpus heterophyllus Lam.) -- Somatic Embryogenesis in Indian Olive (Elaeocarpus robustus L) -- Rescue of Endangered Palms by in vitro Methods: The Case of ‘Bottle Palm’ -- Somatic Embryogenesis in American Grapes (Vitis x labruscana L.H. Bailey) -- Pistachio (Pistacia vera L.) -- Grape (Vitis vinifera L.) -- Date Palm Phoenix dactylifera L. -- Somatic Embryogenesis Protocol: Citrus -- Olive (Olea europaea L.) -- Section C -- Protocol of Somatic Embryogenesis: Dalbergia sissoo Roxb. (Sissoo) -- Protocol of Somatic Embryogenesis: Pedunculate Oak (Quercus robur L.) and Sessile Oak (Quercus petraea /Matt./ Liebl.) -- Protocol of Somatic Embryogenesis: Tamarillo (Cyphomandra betacea (Cav.) Sendtn.) -- Protocol of Somatic Embryogenesis: European Chestnut (Castanea sativa Mill.) -- Protocol of Somatic Embryogenesis in Acacia arabica (Lamk.) Willd. -- Protocol for Hazelnut Somatic Embryogenesis -- Protocol of Somatic Embryogenesis: Ocotea catharinensis Mez. (Lauraceae) -- Cork Oak, Quercus suber L. -- Sawara Cypress Chamaecyparis pisifera Sieb. et Zucc. -- Protocol of Somatic Embryogenesis: Holm Oak (Quercus ilex L.) -- Protocols for Somatic Embryogenesis of Hybrid Firs -- Somatic Embryogenesis in Sandalwood -- Echinacea purpurea L.: Somatic Embryogenesis from Leaf Explant -- Section D -- Histological Techniques -- Bioencapsulation of Somatic Embryos in Woody Plants -- Protoplast Isolation and Culture of Woody Plants -- Cryopreservation of Embryonal Cells -- Double Staining Technology for Distinguishing Embryogenic Cultures -- Thin Cell Layer Sectioning for Inducing Somatic Embryogenesis in Woody Plants.

Rapidly growing human population, decreasing arable land, combined with environmental degradation are causes of concern to agriculturists in sustaining agriculture production and loss of genetic biodiversity. Plant breeders are under continuous pressure for finding new ways for sustainable agriculture by using natural and induced genetic diversity, which are essential to crop improvement. (Jain 2002a) Plant breeders have successfully recombined the desired genes from cultivated crop germplasm and related species by sexual hybridization, and have also been able to develop new cultivars with desirable traits such as high yield, disease, insect and pests, and drought resistance. No further scope of expanding arable land is foreseen. Moreover, rapid industrialization is mounting a heavy toll on the environment, including atmospheric ozone layer depletion, acid rain, erratic weather conditions, insect and pests, diseases, global warming and increase of ultra-violet (UV-B) radiationlevel. The adverse environmental impact will gradually lead to water shortage and reduction in crop production. Thereby, plant breeders need to adopt both conventional and new technologies such as induced mutations, plant tissue culture, and recombinant DNA for sustainable food production in order to feed the world (Jain 2002a).

An efficient plant regeneration system by tissue culture is a prerequiste for the successful use of biotechnology in forest improvement programmes. Plant tissue culture has a great potential for rapid multiplication of elite tree genotypes for the large-scale plant production (Jain, 1997; Ahuja, 1992). This technology is highly labour intensive and cost effective, and is being widely used in the developing countries (Table 1). Since forest trees are long lived and highly heterozygous, it is important that regenerated plants are genetically uniform of the selected genotypes (Klopfenstein et al, 1997). Commonly, all cell and tissue culture techniques require the growth of tissue and organ culture segments on a suitable nutrient medium that stimulate the development of either callus or direct shoot formation or organogenesis. The regenerated shoots are rooted either in vitro or in vivo. This approach of plant regneration is termed as micropropagation, which is commercially used for large-scale plant multiplication (Jain, 1997).

Plant genetic diversity is highly essential for the genetic improvement of crops for sustainableagriculture and its gradual loss is as a consequence to rapid human population growth, industrialization,deforestation, and natural calamities. In the future, the impact of climate change may have an adverseimpact on sustainable date palm productions as well other crops. The conservation, distribution and properutilization of plant genetic diversity/resources become necessary for the development and improvement ofdate palm cultivars for sustainable crop production. The conservation, distribution, and utilization ofnatural and induced genetic diversity have become essential by the establishment of gene/germplasm bankboth at the national and international levels. The Gene bank should encourage researchers to survey andmonitor the genetic diversity of natural populations and landraces on farmer's fields. In vitro conservationtechniques, cryopreservation or cryo-storage and cold storage, are excellent system for genetic resourcesconservation of forest trees and horticultural crops. Cold-storage approach has disadvantage of frequentsubculture and that may run into a risk of contamination and somaclonal variation. Cryo-storage has anadvantage of long-term storage without going through frequent subcultures and somaclonal variation. Forthis, in vitro cultures are suitable, e.g. somatic embryos/ cell suspension, callus, and should be able toregenerate plants with minimal somaclonal variation. In date palm, the most common in vitro cultureapproach has been somatic embryogenesis, which is very much dependent on genotype and culturemedium for plant multiplication, even though there is a risk of genetic variability among regenerated plantsFor the first time, cryo-storage of date palm somatic embryos was done in Tunisia, FAO/IAEA project, andplant regeneration is yet to be accomplished. In Asia, National Bureau of Plant Genetic Resources(NBPGRI, India) is the biggest germplasm bank, and conserves mainly local germplasm seed andvegetative propagated crops and introduces new crops as well.

The aim of this study was to isolate Phytopthora cactorum-resistant strawberry plants, regenerated from gamma-irradiated explants on a shoot regeneration medium. Three gamma doses (0, 5, 10, 15 krad) were used to irradiate strawberry axillary buds taken from in vitro-grown plants. After irradiation, axillary buds were cultured on a shoot regeneration medium containing 0.75 mg BA/liter and 0.4 mg IBA/liter. Shoot regeneration occurred mainly from axillary buds irradiated with 5 and 10 krad. The highest dose (15 krad) produced few shoots. The shoot regeneration rate was highest at the 50-krad dose. All the regenerated plants were transferred in the greenhouse. The crude extract of P. cactorum, isolated from the strawberry field, was prepared in sterile water; 1 ml of it was put directly in the center of the crown of each of 400 regenerated plants. After 2 weeks, leaves of most of the plants wilted. Only 20 plants survived the first round of selection; they grew slowly when compared with the control and also showed some tolerance to drought. Further investigations are in progress to reconfirm the resistance of selected putative disease-resistant strawberry plants.

Embryogenic callus development was initiated in cultured immature embryo explants of four different genotypes of slash pine (Pinus elliottii) on several culture media supplemented with auxin and cytokinin. Callus induction, either from the suspensor cells at the base of immature embryos or from the cotyledons of immature embryos, started after 1–2 weeks. As the callus started to proliferate, three types of calli were distinguished; white mucilaginous, loose globular, and light green-loose. Histochemical staining, with acetocarmine, indicated that only white mucilaginous calli were embryogenic. Non-embryogenic loose globular and light green-loose calli continued to retain their nature even after several subcultures. The origin of somatic proembryos in Pinus elliottii was from single cells of the suspensor cell callus. The developmental stage of the zygotic immature embryos, genotype and culture medium are critical factors for the production of embryogenic callus. Somatic proembryos are being cultured for further development.