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37 p.-8 fig., Somatic embryogenesis is considered a convenient tool for investigating the regulating mechanisms of embryo formation; it is also a feasible system for in vitro regeneration procedures, with many advantages in woody species. Nevertheless, trees have shown recalcitrance to somatic embryogenesis, and its efficiency remains very low in many cases. Consequently, despite the clear potential of somatic embryogenesis in tree breeding programs, its application is limited since factors responsible for embryogenesis initiation have not yet been completely elucidated. In the present work, we investigated key cellular factors involved in the change of developmental program during leaf somatic embryogenesis initiation of white oak (Quercus alba), aiming to identify early markers of the process. The results revealed that pectin esterification, auxin accumulation and DNA demethylation were induced during embryogenesis initiation and differentially found in embryogenic cells, while they were not present in leaf cells before induction or in non-embryogenic cells after embryogenesis initiation. These three factors constitute early markers of leaf embryogenesis and represent processes that could be interconnected and involved in the regulation of cell reprogramming and embryogenesis initiation. These findings provide new insights into the mechanisms underlying plant cell reprogramming, totipotency and embryogenic competence acquisition, especially in tree species for which information is scarce, thus opening up the possibility of efficient manipulation of somatic embryogenesis induction., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (ERDF/FEDER) [grant numbers AGL2014-52028-R and AGL2013-47400-C4-3-R].

Cryopreservation of selected genotypes of European chestnut and cork oak was carried out in two laboratories in a project involving conservation of field collections. Plant material was selected on the basis of disease resistance (chestnut), growth habit, phytosanitary performance and cork quality (cork oak). The cryopreservation technique comprised of vitrification of shoot apices isolated from in vitro stock shoot cultures (chestnut) and somatic embryos (cork oak). Forty-three out of 46 chestnut genotypes assayed survived the freezing process, but only 63% recovered their capacity to produce new shoots. After completion of multiplication and rooting steps, the surviving shoots produced plants that were morphologically identical to those derived from non-supercooled material. All 51 cork oak genotypes withstood freezing and were able to produce new somatic embryos through a process of secondary embryogenesis. Multiplication and germination of the recovered embryos enabled production of plants that were morphologically identical to those derived from non-supercooled material. In light of the results obtained, long-term cryopreservation of these species is feasible, thereby ensuring conservation of valuable genotypes during field evaluation.

For the mass production of chestnut trees with selected, hybrid, or genetically engineered genotypes, one potentially desirable propagation strategy is based on somatic embryogenesis. Although methods exist for the initiation of embryogenic cultures of Castanea sativa from immature zygotic embryos or leaf explants, the embryos produced have had low rates of conversion into plantlets. This study explored the possible benefits for somatic embryos that have already undergone maturation and cold treatments, of (a) partial slow or fast desiccation, and (b) of the addition of plant growth regulators or glutamine to the germination medium. Germination response was evaluated in terms of both conversions to plantlets and through embryos developing only shoots (shoot germination) that could be rooted following the micropropagation protocols developed for chestnut. Two or 3 wk slow desiccation in sealed empty Petri dishes resulted in a slight reduction in water content that nevertheless increased total potential plant recovery, shoot length, and the number of leaves per plantlet. However, best results were achieved by 2 h fast drying in a laminar flow hood, which reduced embryo moisture content to 57–58% and enhanced the potential plant recovery and quality of regenerated plantlets. Plant yield was also promoted by addition of 0.44 μM benzyladenine and 200–438 mg/l of glutamine to the germination medium, and plantlet quality (as evidenced by root, shoot, and leaf growth) by the further addition of 0.49 μM indole-3-butyric acid., The study was funded by the Ministerio de Educación y Ciencia (Spain) through the project AGL2005-00709.

Experiments were performed to determine the influence of maturation medium carbohydrate content on the rates of germination and plantlet conversion (root and shoot growth) of somatic embryos from four embryogenic lines derived from leaf or internode explants of Quercus robur L. seedlings. The conversion rate was favoured by high carbohydrate content as long as the maturation medium contained at least 2 percnt; sucrose, which was necessary for healthy embryo development. Given this, sorbitol and mannitol favoured the conversion rate more efficiently than sucrose, the highest rate, 32 percnt;, being achieved by medium with 6 percnt; sorbitol and 3 percnt; sucrose. Maturation treatment did not affect the root or shoot lengths of converted embryos. In supplementary experiments, 2 weeks of gibberellic acid treatment between maturation and germination treatments did not improve germination rates, but did reduce root length and the number of leaves per regenerated plantlet. In the four embryogenic lines tested, plant recovery rate was enhanced by inclusion of benzyladenine into the germination medium following culture of the embryos on maturation medium with 6 percnt; sorbitol and 2-3 percnt; sucrose. In embryogenic systems it is important to assess the uniformity of the regenerants. Random amplified polymorphic DNA (RAPD) analysis using 32 arbitrary oligonucleotide primers was performed to study variability in DNA sequences within and between four embryogenic lines. No intraclonal nor interclonal polymorphism was detected between embryogenic lines originating from different types of explant from the same seedling, but every one of the primers detected enough polymorphism among clones originating from different plants to allow these three origins to be distinguished. No differences in DNA sequences between regenerated plantlets and their somatic embryos of origin were detected, but a nodular callus line that had lost its embryogenic capacity was found to be mutant with respect to three other clones originating from the same plantlet. This study shows that high carbohydrate levels in the maturation medium significantly increase plant conversion of oak somatic embryos, which exhibit no variation in DNA sequences when proliferated by secondary embryogenesis., This research was partially supported by CYCIT, Spain (AGL 2000–0297-C03–02) and Xunta de Galicia, Spain (PGIDT 00BIO 40001).

Development of a system for direct transfer of antifungal candidate genes into European chestnut (Castanea sativa) would provide an alternative approach to conventional breeding for production of chestnut trees that are tolerant to ink disease caused by Phytophthora spp. Overexpression of genes encoding PR proteins (such as thaumatin-like proteins), which display antifungal activity, may represent an important advance in control of the disease. We have used a chestnut thaumatin-like protein gene (CsTL1) isolated from European chestnut cotyledons and have achieved overexpression of the gene in chestnut somatic embryogenic lines used as target material. We have also acclimatized the transgenic plants and grown them on in the greenhouse. Here, we describe the various steps of the process, from the induction of somatic embryogenesis to the production of transgenic plants.

In somatic embryogenic systems of various oak species, including Quercus alba and Q. rubra, embryogenic lines generally proliferate through generation of nodular embryogenic structures (NS) as a form of repetitive embryogenesis, without embryo development progressing to the cotyledonary stage, or with a low proportion of embryo formation, resulting in the inability to achieve suitable material for plant recovery. In experiments we used previously initiated embryogenic lines derived from 7-year-old trees of Q. alba and Q. rubra. The aim of this work was to improve embryo proliferation and histodifferentiation of lines from these oak species to obtain well-developed embryos and plant regeneration. The effects of activated charcoal, silver thiosulphate (STS) and sucrose concentration on secondary embryo production were investigated. Overall, the best embryo proliferation medium consisted of basal Murashige and Skoog medium containing 0.4 % charcoal and 20 µM STS, and supplemented with 6 % sucrose for Q. alba and 3 % sucrose for Q. rubra. In these conditions, well-developed and singularised torpedo- and cotyledonary-shaped embryos were produced. The developmental stage of the embryogenic explant used for subculture significantly influenced embryo production and differentiation of new secondary embryos, with NS being the most effective explant despite being the smallest explant type in terms of size and fresh weight. Addition of STS in the germination medium also had a positive effect on germination response giving rise to approximately 39 % embryo conversion in Q. alba and 11 % in Q. rubra., This research was partially funded by Ministerio de Economía y Competitividad (Spain) through the project AGL2013-47400-C4-3-R.

Development of a system for direct transfer of antifungal candidate genes into European chestnut (Castanea sativa) would provide an alternative approach to conventional breeding for production of chestnut trees that are tolerant to ink disease caused by Phytophthora spp. Overexpression of genes encoding PR proteins (such as thaumatin-like proteins), which display antifungal activity, may represent an important advance in control of the disease. We have used a chestnut thaumatin-like protein gene (CsTL1) isolated from European chestnut cotyledons and have achieved overexpression of the gene in chestnut somatic embryogenic lines used as target material. We have also acclimatized the transgenic plants and grown them on in the greenhouse. Here, we describe the various steps of the process, from the induction of somatic embryogenesis to the production of transgenic plants.

Adventitious shoots were induced on the proximal portion of leaves excised from Fagus orientalis shoot cultures derived from a 2-mo.-old or a 4-yr-old seedling. Up to 90% of the explants formed between 13 and 19 buds after culture on Woody Plant Medium containing 2.9 μM indole-3-acetic acid and 4.5 μM thidiazuron. Adventitious buds developed mostly on petiole stub callus, but also on the midvein. The histological events leading to shoot organogenesis were examined. Some shoots developed directly from subepidermis or epidermis, but most originated indirectly from cell file proliferation produced by periclinally dividing cells subadjacent to the epidermis. Some cells in the outermost layers of these files became meristematic and divided extensively, resulting in the formation of meristemoids after 16 d of culture. Dedifferentiation into meristematic cells, which exhibited a large, prominent nucleus, densely-stained cytoplasm, and a high nucleus-to-cell area ratio, was generally associated with anticlinal divisions in cells previously originated by periclinal division. Subepidermal cell proliferation occurred mainly in the abaxial surface of the explant, which initially formed a diffuse cambium and later evolved to a phellogenic cambium. Some meristemoids were also differentiated by lenticel phellogen. Organized cell divisions in meristemoids gave rise to bud primordia that emerged from the explant surface and differentiated a protoderm. The progressive structural differentiation of the apical meristem, leaf primordia, and procambial strands led, after about 28 d of culture, to shoots with vascular connections with treachery elements previously differentiated in adjacent tissues.

Somatic embryos of Camellia japonica were hydrogel encapsulated using 3% sodium alginate and 0.1 M calcium chloride to produce synthetic seeds. Both germinability and repetitive embryogenesis capacity of the encapsulated embryos were investigated. The frequency of in vitro germination into plants of artificial seeds was affected by various nutrient additives included in the encapsulating matrix. The addition of Ca-free Murashige and Skoog basal medium plus 3% sucrose plus 14.4 µM gibberellic acid and 28.5 µM indole-3-acetic acid to the alginate capsule resulted in 63% plant recovery rate, which was similar to that of non-encapsulated embryos. Encapsulation of somatic embryos did not negatively affect the maintenance of their embryogenic competence, the mean number of secondary embryos being significantly increased when the alginate beads included the growth regulators of the secondary embryogenesis medium (4.44 µM 6-benzyladenine and 0.41 µM indole-3-butyric acid). Storage at 4°C significantly reduced the survival and germination into plants frequencies of both encapsulated and non-encapsulated embryos, but the reduction was much greater for non-encapsulated embryos. Plant recovery of encapsulated embryos was 40% and 30% following storage for 30 and 60 days, respectively.

Adventitious shoots were induced on transversally divided expanding leaves fromFagus sylvatica shoot cultures of juvenile origin. Adventitious shoot buds formed mainly on callus that developed on the petiole stump or on the cut across the midrib of distal leaf halves. However, sometimes they arose directly from leaf tissue. An anatomical study confirmed both the direct and indirect origin of the adventitious buds. The best results were obtained by culturing proximal leaf sections on woody plant medium supplemented with 2.9 μM indole-3 acetic acid in combination with 8.9 μM benzyladenine or 2.3 μM thidiazuron (TDZ). Proximal explants were more responsive than distal explants in terms of both callus formation and bud regeneration, regardless of the induction medium or clone tested. Bud formation capacity was influenced by the genotype of the stock shoot culture and was enhanced by an initial 10 d darkness, but was inhibited by longer periods of darkness. Caulogenic competence was significantly affected by the duration of exposure to TDZ; in particular, adventitious shoot length was depressed by increasing the exposure period. Three weeks culture with TDZ was the most efficient treatment for shoot production and elongation. Further shoot development was promoted by subculturing the explants to the same medium used for the maintenance of the stock shoot cultures. Shoots so obtained were multiplied and rooted producing plantlets of adventitious origin.

Cryopreservation in liquid nitrogen was attempted with both somatic embryos and zygotic embryonic axes of the ornamental Camellia japonica L. Several protective measures were applied to somatic embryos (desiccation, chemical protectors, hardening by culture at low temperatures, encapsulation in alginate beads), but none allowed somatic embryos cultures to survive after 24 h in liquid nitrogen. Embryonic axes, however, were easily cryopreserved by means of the simplest technique: desiccation in a laminar flow hood and direct immersion in liquid nitrogen. Although the causes of the difference in cryopreservability between the two types of material are not known, one might be the difference between their degrees of differentiation and water content.

Somatic embryogenesis is a powerful tool for plant regeneration and also provides a suitable material for investigating the molecular events that control the induction and development of somatic embryos. This study focuses on expression analysis of the QrCPE gene (which encodes a glycine rich protein) during the initiation of oak somatic embryos from leaf explants and also during the histodifferentiation of somatic embryos. Northen blot and in situ hybridization were used to determine the specific localization of QrCPE mRNA. The results showed that the QrCPE gene is developmentally regulated during the histodifferentiation of somatic embryos and that its expression is tissue- and genotype- dependent. QrCPE was strongly expressed in embryogenic cell aggregates and in embryogenic nodular structures originated in leaf explants as well as in the protodermis of somatic embryos from which new embryos are generated by secondary embryogenesis. This suggests a role for the gene during the induction of somatic embryos and in the maintenance of embryogenic competence. The QrCPE gene 2 was highly expressed in actively dividing cells during embryo development, suggesting that it participates in embryo histodifferentiation. The localized expression in the root cap initial cells of cotyledonary somatic embryos and in the root cap of somatic seedlings also suggests that the gene may be involved the fate of root cap cells.

The availability of a system for direct transfer of antifungal candidate genes into European chestnut (Castanea sativa Mill.) would offer an alternative approach to conventional breeding for production of chestnut trees tolerant to ink disease caused by Phytophthora spp. For the first time, a chestnut thaumatin-like protein gene (CsTL1), isolated from chestnut cotyledons, has been overexpressed in three chestnut somatic embryogenic lines. Transformation experiments have been performed using an Agrobacterium tumefaciens Smith and Townsend vector harboring the neomycin phosphotransferase (NPTII) selectable and the green fluorescent protein (EGFP) reporter genes. The transformation efficiency, determined on the basis of the fluorescence of surviving explants, was clearly genotype dependent and ranged from 32.5% in the CI-9 line to 7.1% in the CI-3 line. A total of 126 independent transformed lines were obtained. The presence and integration of chestnut CsTL1 in genomic DNA was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. Quantitative real-time PCR revealed that CsTL1 expression was up to 13.5-fold higher in a transgenic line compared with its corresponding untransformed line. In only one of the 11 transformed lines tested, expression of the CsTL1 was lower than the control. The remaining 115 transformed lines were successfully subjected to cryopreservation. Embryo proliferation was achieved in all of the transgenic lines regenerated and the transformed lines showed a higher mean number of cotyledonary stage embryos and total number of embryos per embryo clump than their corresponding untransformed lines. Transgenic plants were regenerated after maturation and germination of transformed somatic embryos. Furthermore, due to the low plantlet conversion achieved, axillary shoot proliferation cultures were established from partially germinated embryos (only shoot development), which were multiplied and rooted according to procedures already established. Transgenic plants were acclimatized and grown in a greenhouse. No phenotypic differences were found with control plants, suggesting no potential cytotoxic effects of the green fluorescent protein. The results reported in the present work could be considered as a first step toward the production of fungal-disease tolerant cisgenic chestnut plants., This research was partially funded by Xunta de Galicia (Spain) through the project 09MRU002400PR.

Theoretically, complete rejuvenation of mature trees should occur through somatic embryogenesis, however, this has not been extensively studied. The main objective of the present study was to increase the efficiency of in vitro clonal propagation for mature Quercus robur (100–300 years old), by induction of somatic embryogenesis as rejuvenation step prior to establishment of shoot culture through micropropagation of somatic embryo-derived plantlets. Shoot culture lines of “mature” origin were established from epicormic shoots of two centenarian oak genotypes (Sainza and CR-0) and maintained by axillary shoot proliferation. Embryogenic lines were also initiated from epicormic leaf explants of the same genotypes and maintained by secondary somatic embryogenesis. Although the frequency of somatic embryo conversion into plantlets was low in pedunculate oak, shoot culture lines could be established and maintained by axillary branching from several germinated somatic embryos. For each genotype and shoot culture line of the two origins (mature tree and somatic plantlets), shoot multiplication rate and elongation as well as rooting ability parameters were compared. Compared with “mature-origin” shoot cultures and after more than one year propagation in vitro, shoot lines established from somatic plantlets produced a significantly higher proportion of elongated, rootable shoots (from 26.0–31.6 to 36.8–40.5%) with increased rooting ability (from 3.3–45.6% to 23.2–89.8%). In the case of 300-year-old Sainza genotype such a high organogenic capacity was similar to shoot cultures initiated from basal sprouts. Basal sprouts are considered as “mature” material that retains juvenile characteristics compared with epicormic shoots forced from crown branches. Somatic embryogenesis only slightly improved plant regeneration of shoot cultures from basal sprouts, thus validating their use as “juvenile control”. The present results provide evidence that some rejuvenation occurred during the process of somatic embryogenesis and resulted in improved shoot growth and rooting of somatic embryo-derived culture compared with “mature” shoot culture. The results reported in this study might be useful in embryogenic systems with low plant conversion rates. The proposed experimental model might also be useful in finding molecular markers of plant ontogeny., This research was partially funded by Xunta de Galicia and Consejo Superior de Investigaciones Científicas (Spain) through the projects 09MRU002400PR and PIE200940I011, respectively.

The ability of plants to remove pollutants from the environment is currently used in a simple and low-cost cleaning technology known as phytoremediation. Unfortunately, little is known about the metabolic pathways involved in the transformation of xenobiotic compounds and the ability of certain plants to tolerate, detoxify, and store high concentrations of heavy metals. Plant cell and tissue culture is considered an important tool for fundamental studies that provide information about the plant-contaminant relationships, help to predict plant responses to environmental contaminants, and improve the design of plants with enhanced characteristics for phytoremediation. Callus, cell suspensions, hairy roots, and shoot multiplication cultures are used to study the interactions between plants and pollutants under aseptic conditions. Many plant species have an inherent ability to accumulate/metabolize a variety of pollutants, but they normally produce little biomass. However, fast-growing trees are excellent candidates for phytoremediation because of their rapid growth, extensive root system, and high water uptake. This chapter outlines the in vitro plant production of both somaclonal variants and transgenic plants of Populus spp. that exhibit high tolerance to heavy metals.

The genus Quercus, which belongs to the family Fagaceae, is native to the northern hemisphere and includes deciduous and evergreen species. The trees of the different species are very important from both economic and ecological perspectives. Application of new technological approaches (which span the fields of plant developmental biology, genetic transformation, conservation of elite germplasm and discovery of genes associated with complex multigenic traits) to these long-rotation hardwoods may be of interest for accelerating tree improvement programs. This review provides a summary of the advances made in the application of biotechnological tools to specific oak species. Significant progress has been made in the area of clonal propagation via organogenesis and somatic embryogenesis (SE). Standardized procedures have been developed for micropropagating the most important European (Q. robur, Q. petarea, Q. suber) and American (Q. alba, Q. bicolor, Q. rubra) oaks by axillary shoot growth. Although regenerated plantlets are grown in experimental trials, large scale propagation of oak species has not been carried out. The induction of SE in oaks from juvenile explants is generally not problematic, although the use of explants other than zygotic embryos is much less efficient. During the last decade, enormous advances have been made in inducing SE from selected adult trees, mainly specimens of pedunculate oak (Q. robur) and cork oak (Q. suber). Advances in the understanding of the maturation and germination steps are required for better use of embryogenic process in clonal forestry. Quercus species are late-maturing and late-flowering, exhibit irregular seed set, and produce seeds that are recalcitrant to storage by conventional procedures. Vitrification-based cryopreservation techniques were used successfully in somatic embryos of pedunculate oak and cork oak and an applied genbank of cork oak selected genotypes is now under development. The feasibility of genetic transformation of pedunculate oak and cork oak somatic embryos by means of co-culture techniques with several strains of Agrobacterium tumefaciens has also been demonstrated.To date, most research on the genomics of Quercus species has concerned population genetics. Approaches using functional genomics to examine the molecular and cellular mechanisms that control organogenesis and or somatic embryogenesis are still scarce and efforts on the isolation and characterization of genes related to other specific traits should be intensified in the near future, as this would help improve the practical application of clonal forestry in recalcitrant species such as oaks., Xunta de Galicia (Spain), proyecto: 09MRU002400PR.

The ability of plants to remove pollutants from the environment is currently used in a simple and low-cost cleaning technology known as phytoremediation. Unfortunately, little is known about the metabolic pathways involved in the transformation of xenobiotic compounds and the ability of certain plants to tolerate, detoxify, and store high concentrations of heavy metals. Plant cell and tissue culture is considered an important tool for fundamental studies that provide information about the plant-contaminant relationships, help to predict plant responses to environmental contaminants, and improve the design of plants with enhanced characteristics for phytoremediation. Callus, cell suspensions, hairy roots, and shoot multiplication cultures are used to study the interactions between plants and pollutants under aseptic conditions. Many plant species have an inherent ability to accumulate/metabolize a variety of pollutants, but they normally produce little biomass. However, fast-growing trees are excellent candidates for phytoremediation because of their rapid growth, extensive root system, and high water uptake. This chapter outlines the in vitro plant production of both somaclonal variants and transgenic plants of Populus spp. that exhibit high tolerance to heavy metals.

The genus Castanea, chestnuts and chinkapins, belongs to the family Fagaceae, which includes other important timber producing genera such as Quercus and Fagus. The genus Castanea is divided into three geographically delimited sections with at least seven consistently recognized interfertile species: 4 species in Asia (C. mollissima, C. henryi, C. seguinii, and C. crenata), two or more species in North America (C. dentata, C. ozarkensis, and C. pumila) and one in Europe and Turkey (C. sativa). The two most important diseases of chestnut are ink disease (Phytophthora) and chestnut blight (Cryphonectria). Resistance to these is the major objective for rootstock breeding in Europe and scion breeding in North America. In both cases, the source of resistance was Asian species. European breeding programs developed resistant hybrid rootstocks, which are propagated by stooling, cuttings, or in vitro culture. A major pest of chestnut is the gall wasp Dryocosmus kuriphilus whose control is based on the spread of parasitoids but also on the selection of resistant cultivars. For nut production, the most important breeding objectives include the following: good horticultural traits, product quality, suitability to storage and processing, and ease of peeling. For timber, important characters include wood quality, rapid growth, and nonchecking of wood (ring-shake). Molecular maps have been developed, which has expanded the genetic knowledge of the chestnut. An efficient genetic transformation protocol for C. sativa through the coculture of somatic embryos with different strains of Agrobacterium tumefaciens has been described.

For Castanea sativa (European chestnut), a species with recalcitrant seeds that is not easily propagated vegetatively, cryopreservation is one of the most promising techniques for maintaining genetic resource diversity and for conservation of selected germplasms. Long-term conservation of selected seeds and valuable embryogenic lines can be achieved through the cryopreservation of zygotic embryonic axes and somatic embryos, respectively. This chapter describes methods for the desiccation-based cryostorage of zygotic embryonic axes, and the vitrification-based cryopreservation of somatic embryos. For zygotic embryonic axes, the highest post-thaw survival and plantlet recovery rates are obtained by desiccation in a laminar flow hood to 20-25% moisture content, followed by direct immersion in liquid nitrogen. For somatic embryos, embryogenesis resumption rates of over 60% are achieved by preculture of embryo clumps for 3 days on solid medium containing 0.3 M sucrose, incubation in PVS2 vitrification solution for 60 min at 0°C, and direct immersion in liquid nitrogen. Plantlet recovery from cryostored embryogenic lines requires proliferation of the thawed embryos and subsequent maturation before germination and conversion into plantlets.

An in vitro shoot multiplication system was established from juvenile Fagus sylvatica L. tissues, and plantlets were regenerated. Embryonic axes were excised from beech seeds and germinated in vitro on media supplemented with 6-benzyladenine (BA) to obtain plantlets with axillary shoots. Shoot multiplication was maintained by sequential subculture of axillary shoot tips and basal segments on Woody Plant Medium supplemented with 0.5 mg/liter BA+2 mg/liter zeatin+0.2 mg/liter naphthaleneacetic acid (NAA). The effeciency of shoot multiplication clearly depended on the kind of explant used. Transfer to fresh medium every 2 wk during the 6-wk multiplication cycle improved multiplication rates. In the rooting stage, an initial 7-day dark period significantly improved rooting capacity and accelerated the emergence of roots on auxin-treated shoots. Adventitious buds were induced on the intact hypocotyls of the whole plantlets derived from the initial embryonic axis explants, especially on those cultured on medium with 1 mg/liter BA. Cotyledon and hypocotyl segments isolated from seedlings grown in vitro from embryos also exhibited capacity for adventitious bud formation, especially when cultured on media supplemented with 0.5 mg/liter BA + 0.1 mg/liter NAA.

This article describes the induction of caulogenesis and somatic embryogenesis in juvenile leaf fragments of Camellia japonica and Camellia reticulata cultivar 'Mouchang'. In C. japonica cultivar 'Alba Plena' adult leaves, only callus formation occurred. Light inhibited both callus formation and morphogenesis; initial dark period of at least 14 weeks was required for bud differentiation and somatic embryogenesis. Best results were obtained using Murashige and Skoog's medium supplemented with 35.5 μM 6-benzyladenine + 2.5 μM indole-3-butyric acid. Somatic embryos and adventitious buds were successfully multiplied, and whole plantlets regenerated. For C. japonica there was a significant difference in rooting rate between shoots of axillary and adventitious origin; for C. reticulata, axillary and adventitious shoots did not differ in rooting rate, but did differ significantly in root length and the number of roots per shoot.

A protocol was developed for genetic transformation of somatic embryos derived from juvenile and mature Quercus robur trees. Optimal transformation conditions were evaluated on the basis of the results of transient GUS expression assays with five oak embryogenic lines and a strain of Agrobacterium tumefaciens (EHA105) harbouring a p35SGUSINT plasmid containing a nptII and a uidA (GUS) genes. For stable transformation, embryo clumps at globular/torpedo stages (4–10 mg) were inoculated with EHA105:p35SGUSINT bacterial cultures, cocultivated for 4 days and selected in proliferation medium with 75 mg/l of kanamycin. Putatively transformed masses appeared after 20–30 weeks of serial transfers to selective medium. Histochemical and molecular analysis (PCR and Southern blot) confirmed the presence of nptII and uidA genes in the plant genomes. Transformation efficiencies ranged from up to 2% in an embryogenic line derived from a 300-year-old tree, to 6% in a juvenile genotype. Twelve independent transgenic lines were obtained from these oak genotypes, and transgenic plantlets were recovered and acclimatized into the soil. This is the first demonstration of the production of transformed somatic embryos and regenerated plants from juvenile and mature trees of Q. robur and suggests the possibility of introducing other genetic constructions to develop trees that are tolerant/resistant to pathogens and/or biotic stresses., This research was partially funded by the Ministerio de Educación y Ciencia and Xunta de Galicia (Spain) through projects AGL2006-01387/FOR and 09MRU002400PR, respectively.

An anatomical study was carried out during the sequences of events which lead to the differentiation of secondary embryos of Camellia reticulata cv 'Mouchang'. Secondary embryogenesis can be induced by culturing somatic embryos on a modified Murashige and Skoog medium supplemented with 0.5 mg·liter-1 6-benzylaminopurine and 0.1 mg·liter-1 indole-3-butyric acid. After about 12 days of culture, globular-shaped secondary embryos became apparent, and by 18 to 20 days of culture cotyledonary stages were formed. Embryos developed mainly on the hypocotyl of primary embryos without an intermediate callus. Histologic monitoring revealed that secondary embryos apparently had a multicellular origin from embryogenic areas originating in both epidermal and subepidermal layers of the hypocotyl region. This morphogenetic competence is related to the presence, at the time of culture, of relatively undifferentiated cells in superfical layers of the primary embryo hypocotyl. Microcomputer image analysis was applied for quantifying cytological events associated with somatic embryogenesis. This method showed an increasing gradient in the nucleus-to-cell area ratio from differentiated cells passing through preembryogenic cells to embryogenic cells. The formation of embryogenic areas was preceded by accumulation of starch in the surrounding cortical cells. The cells underlying globular secondary embryos still contained abundant starch, but it declined as the secondary embryos developed

Somatic embryos were induced on the roots of Camellia japonica L. plantlets regenerated from an in vitro clone of juvenile origin. The embryos appeared to differentiate from epidermic cells and to be connected with the root via a few parenchymatous cells. Somatic embryogenesis occurred on basal medium and with or without various combinations of zeatin, BA, and IBA. Secondary embryos were induced on cotyledons and/or hypocotyl regions of somatic embryos. Two morphological types of somatic embryos were developed, seed-like and bud-like types, and their formation was influenced by the presence of BA in the medium. Embryogenic capacity has been maintained for more than 24 months by subculturing secondary embryos at 7- to 8-week intervals. The best gibberellin/auxin combination for inducing the germination of isolated somatic embryos was GA at 5 mg·liter-1 G A3 and IAA at 1 mg·liter-1. P1antlets were successfully established in planting medium and have continued to grow in a greenhouse. Chemical names used: N-(phenylmethyl)-1H-purine-6-amine (BA); (1α, 2β, 4aα, 4bβ, 10β)-2,4a,7-trihydroxy-l-methyl-8-methylenegibb-3-ene-1,10-dicarboxylic acid l,4a-lactone (GA); 1 H -indole-3-acetic acid (IAA); 1 H- indole-3-butyric acid (IBA); 2-methyl-4-(1 H- purine-6-ylamino)-2-buten-l-ol (zeatin).

SummaryEffects of various factors were studied on the in vitro multiplication and rooting of a clone of Camellia reticulata cv. Captain Rawes established in vitro from shoots of an adult tree. Multiplication rates were increased by repeated harvesting of cultures in which shoots were subcultured in a horizontal rather than a vertical position. Best rooting responses were obtained with 175.3 mM sucrose in the rooting medium using 8 week old shoots cultured in 500 ml glass jars; these shoots contained more anthocyanin than those from test-tube cultures. The orientation and repeated harvesting of the mother shoots had no effect on rooting response.

Somatic embryos of Quercus robur L. belonging to lines derived from mature trees were germinated in experiments designed to determine the effects of pregermination chilling and the temporary inclusion of thidiazuron (TDZ) in the germination medium. Germination response was evaluated in terms of the percentages of embryos exhibiting root-only elongation, shoot-only elongation, or both (conversion to plantlets). Conversion rates depended on genotype and were lower than those of oak zygotic embryonic axes germinated in vitro. Two months’ storage of somatic embryos at 4°C between maturation and germination treatments did not influence their conversion frequency, although root-only germination was promoted in the three embryogenic lines investigated. Addition of 0.01–5.0 µM TDZ to the germination medium for 7 or 18 days induced multiple shoot formation in the epicotyl region of germinating embryos, although the 18-day treatment was detrimental with TDZ concentrations of 0.5–5.0 µM. Exposure to 0.05–0.1 µM TDZ for 7 days increased the frequency of shoot-only elongation and decreased that of root-only elongation in embryogenic lines with both high and low conversion ability, but whereas the conversion rate of the hard-to-convert line Sainza was significantly increased, that of the readily converted line B-17 was negatively affected. As a result, with 0.1 µM TDZ the total potential plant recovery of Sainza (the conversion rate plus the percentage of embryos exhibiting shoot-only germination) was 64%, approximately the same as that of B-17. Shoots excised from shoot-only germinating embryos can be elongated and rooted by proven micropropagation procedures. An additional result was the finding that adventitious bud regeneration was induced on the cotyledons of TDZ treated embryos (69% with 0.5 µM TDZ), which may prove useful for genetic transformation. This study is the first to have shown that TDZ can increase the germination efficiency of oak somatic embryos., This research was partially supported by DGI, MEC (Spain) through the project AGL2006-01387/FOR.

Camellia reticulata cultivar ‘Captain Rawes’ has been established and multiplied in vitro starting from material taken from the crown of a 90-year-old tree. Multiplication rates depended on the kind of explant used for multiplying subcultures, which was applied every 8 weeks and for which the most suitable macronutrients were those of Woody Plant Medium. In both initiation and multiplication stages, the combination of growth regulators employed was 2 mg l−1 6-benzylaminopurine, 2 mg l−1 zeatin, 2 mg l−1N6-(Δ2-isopentenyl)-adenine and 0.01 mg l−1 indole-3-butyric acid (IBA). Culturing shoots in a horizontal position increased multiplication rates and it was possible for mother shoots so cultured to be re-cultured on fresh medium several times. Rooting rates of 33% were achieved among shoots excised from the second crop of horizontally cultured mother-shoots, that had been kept in the dark for 10 days, by dipping their basal ends in 1 g l−1 IBA solution for 30 min and keeping them in the dark for a further 18 days in Woody Plant Medium with half-strength macronutrients and no growth regulators.

Camellia reticulata L. plantlets were regenerated by direct and indirect somatic embryogenesis from immature zygotic embryos. Initial explants (cotyledon sections and embryonic axes) produced somatic embryos without intermediate callus tissue when grown on Murashige and Skoog’s basal medium with 30 gl-1 sucrose and no growth regulators; the somatic embryos completed their development in 4-6 weeks in the same medium. Embryogenie competence was increased by 0.5 and 1 mg l-1 IBA. Histological observation showed the embryos to originate from epidermal and subepidermal cells of the cotyledon and hypocotyl explants. Secondary somatic embryos developed directly from the cotyledons and hypocotyl region of primary somatic embryos by a process that was morphologically very similar to that occurring on zygotic explants. Direct repetitive embryogenesis was maintained by this system. Up to 40% germination occurred when mature somatic embryos were isolated and incubated in medium supplemented with 1 mgl-1 GA3 + 1 mgl-1...

Final version of the chapter at: http://dx.doi.org/10.1007/978-1-4020-6352-7_28., Since the 1970s, natural resources the world over have been progressively re-evaluated in accordance with the principles of sustainable agriculture. The European chestnut, Castanea sativa Mill., a hardwood species belonging to the family Fagaceae, has not been unaffected by this process. It is an important resource in many parts of the world because of its economic and environmental role in many agroforestry systems, and in Europe it has been gaining in value as a source of timber and nut production and due to the contribution of chestnut groves to the landscape (Bounous, 2005)., Like the American chestnut C. dentata, C. sativa has been plagued for more than a century by ink disease and chestnut blight, caused by the fungi Phytophthora cinnamomi and Cryphonectria parasitica, respectively. A great deal of research on chestnut focuses on the development of vegetative propagation systems capable of satisfying the demand for elite genotypes that provide both high-quality timber and/or nuts and resistance to these diseases. Since chestnut is a difficult-to-root species, grafting is the most frequent conventional propagation technique, although methods for layering and cutting have recently been improved and are widely used in nurseries to propagate ink-disease-resistant Euro-Japanese hybrids. However, as an alternative to conventional vegetative propagation methods, efforts are being made to establish reliable in vitro regeneration systems that allow clonal propagation. The two major systems are based on embryogenesis or on micropropagation of axillary shoots., Thanks are given to Spanish Ministry of Education and Science (project AGL2005-00709) and Xunta de Galicia, Spain (project PGIDIT03RFO40001PR) for financial support.

In oak species, there is paucity of information on the anatomical changes underlying differentiation of somatic embryos from explants of mature trees. A histological study was undertaken to ascertain the cellular origin and ontogenesis of somatic embryos in leaf cultures from a 100-yr-old Quercus robur tree. Somatic embryogenesis was induced in expanding leaves excised from shoots forced from branch segments, following culture on three successive media containing different concentrations of α-naphthaleneacetic acid and 6-benzylaminopurine. The somatic embryogenesis followed an indirect pathway from a callus tissue formed in the leaf lamina. After 4–6 wk of culture, meristematic cells originated in superficial layers of callus protuberances, but these cells evolved into differentiated vacuolated cells rather than embryos. A subsequent dedifferentiation into embryogenic cells occurred later (9–12 wk of culture) within a dissociating callus. Embryogenic cells exhibited dense protein-rich protoplasm, high nucleoplasmic ratio, and contained small starch grains. Successive divisions of these cells led to the formation of a few-celled proembryos and embryogenic cell clumps within a thick common cell wall, which seemed to have originated unicellularly. However, a multicellular origin of larger embryogenic clumps could not be dismissed; these gave rise to embryonic nodular structures that developed somatic embryos of both uni- and multicellular origin. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were apparent., This research was partially supported by DGI, MEC, and Xunta de Galicia (Spain) through the projects AGL2006-01 387/FOR and PGIDIT03RFO40001PR, respectively

Somatic embryogenesis was induced in expanding leaf explants excised from epicormic shoots forced from branch segments taken at four different times of year from a mature oak (Quercus robur L.). Branch segments 2–4 cm in diameter produced most shoots when collected in March. Somatic embryos were induced on explants derived from branches of all collection dates, although collection in November seemed to afford the best results. Germination and conversion ability of embryos of embryogenic lines derived from six oak trees depended heavily on genotype, conversion rates ranging from 0 to 70%. RAPD analyses found no evidence of genetic variation either within or between the embryogenic lines established from three of these trees, or between these lines and the trees of origin, or between somatic embryo derived plantlets and the trees of origin. The embryogenic system used in this study appears to be suitable for true-to-type clonal propagation of mature oak genotypes., This research was partially supported by Xunta de Galicia (Spain) through the project PGIDIT03RFO40001PR.

Plants of European chestnut (Castanea sativa) have been consistently recovered from cryopreserved in vitro-grown shoot apices by using the vitrification procedure. Factors found to influence the success of cryopreservation include the source of the shoot tips (terminal buds or axillary buds), their size, the duration of exposure to the cryoprotectant solution, and the composition of the post-cryostorage recovery medium. The most efficient protocol for shoot regrowth employed 0.5–1.0 mm shoot tips isolated from 1 cm-long terminal buds that had been excised from 3–5-wk shoot cultures and cold hardened at 4°C for 2 wk. The isolated shoot tips were precultured for 2d at 4°C on solidified Gresshoff and Doy medium (GD) supplemented with 0.2M sucrose, and were then treated for 20 min at room temperature with a loading solution (2M glycerol+0.4M sucrose) and for 120 min at 0°C with a modified PVS2 solution before rapid immersion in liquid nitrogen (LN). After 1 d in LN, rapid rewarming and unloading in 1.2M sucrose solution for 20 min, the shoot tips were plated on recovery medium consisting of GD supplemented with 2.2 μM benzyladenine, 2.9 μM 3-indoleacetic acid, and 0.9 μM zeatin. This protocol achieved 38–54% shoot recovery rates among five chestnut clones (three of juvenile origin and two of mature origin), and in all cases plant regeneration was also obtained., The study was partially supported by Xunta de Galicia, Spain(project PGIDITO3RFO40001PR)

Recent progress in somatic embryogenesis from selected mature trees of Quercus suber, has led to a demand for maintenance of a large number of selected embryogenic lines. To facilitate the management of this material a protocol for the long-term storage of this germplasm should be defined. This study reports on the use of a simple vitrification procedure for the successful cryopreservation of three cork oak embryogenic lines. High embryo recovery levels (88-93 percent) were obtained by first preculturing 2-4 mg clumps of two or three globular embryos on semisolid medium containing 0.3 M sucrose for three days, followed by incubation in PVS2 vitrification solution at 0 degree C for 60 min before direct immersion in liquid nitrogen. The mean number of embryos produced per explant was significantly greater for cryostored embryos than for untreated stock cultures, but the productivity of the latter was recovered in subsequent subcultures of the material produced by cryostored embryos. The germination and plant regeneration rates achieved by cultures derived from cryostored embryos, around 60 percent, were similar to those of non-cryopreserved stock cultures.

This work describes experiments demonstrating the feasibility of long-term conservation of Castanea sativa germplasm through cryopreservation of embryonic axes or somatic embryo clumps. Between 93 % and 100 % of excised embryonic axes of recalcitrant chestnut seeds survived storage in liquid nitrogen (LN) following desiccation in a laminar flow cabinet to moisture contents of 20-24 % (on a fresh weight basis), and some 63 % subsequently developed as whole plants. Desiccation to moisture contents less than 19 % produced damage resulting in loss of organized plant development after cryostorage, allowing only root growth. When 6-8 mg clumps of globular or heart-shaped somatic embryos were precultured for 7 days on high-sucrose medium and then desiccated to a moisture content of 25 % before storage in LN, the embryogenesis resumption level after thawing was 33 %. When the embryo clumps were precultured for 3 days on high-sucrose medium followed by 60 min application of PVS2 vitrification solution before cryostorage, the post-storage embryogenesis resumption level was 68 %.

This study aimed to optimize chestnut micropropagation by defining the best explant type for the shoot multiplication stage and by determining a root induction treatment, to balance rooting rate and plantlet quality, and maximize overall survival after transfer to soil. Shoot cultures of Castanea sativa and C. sativa x C. crenata were established in vitro using shoot tips and nodes from shoots forced on stump sprouts, epicormic shoots and crown branches collected from adult trees. The most productive explant type for subculturing was the basal nodal segment, especially when callus tissue was left on the subcultured basal segment. Although rooting capacity was significantly increased when a 7 d 3 mg 1−1 IBA treatment was combined with an initial 5 d dark period, the latter caused severe shoot necrosis. Acceptable rooting frequencies were achieved by a 24 h, 25–50 mg 1−1 IBA treatments without darkness, and for some clones were enhanced by addition of 1% activated charcoal to the root proliferation medium. Generally, high rooting rates were obtained in charcoal medium when material from the base of the trunk had been used as the source of initial explants. In all clones, charcoal improved lateral root development (as reflected in the total root lengths and the projected root areas measured using a digital image analyser), reduced shoot necrosis symptoms and promoted the reinitiation of shoot growth. The rooting rates achieved in a peat/perlite/vermiculite substrate were similar to those achieved in an charcoal-enriched medium., This research was partially supported by DGICYT, contract PB 92-0017 and by Xunta de Galicia, Spain, contract XUGA 40001 B95.

In vitro shoot cultures of 8 clones or cultivars of Camellia japonica L. and Camellia reticulata Lindley were stored at 2–4 °C for up to 12 months. Survival frequencies near 100% were obtained during the first or second subcultures after cold storage in seven of the eight clones assayed, but the other clone could not be profitably stored for more than 6 months. When shoot tips and nodal expiants of C. japonica L. (clone 2 and cv. Alba Plena) were encapsulated in alginate beads, the regrowth of shoot tips depended on the components of the gel matrix but that of nodal expiants was very poor regardless of the encapsulation conditions. The best morphogenetic response of the shoot tips was obtained when the encapsulating gel was supplemented with the minerals and vitamins of Murashige and Skoog's medium, 3% sucrose and clone-specific growth regulators. The size of the shoot tips used and the concentration of sucrose added to the gel matrix also influenced the capacity for regrowth. Encapsulated shoot tips of C. japonica L. stored at 2–4 °C or 18–20 °C survived for shorter periods of time than unencapsulated shoot tips stored at low temperature: after 75 days of storage, only 10% of encapsulated shoot tips survived at 2–4 °C and 7% at 18–20 °C.

Shoot cultures of chestnut, oak and wild cherry have been stored at low temperature (2°C) for 3, 6, 9 and 12 months. Cultures were stored immediately after the last subculture or 10 d later. Survival and morphogenetic parameters have been recorded at the end of each period of storage. Both survival and proliferation capacity of the explants were influenced by the timing of the transfer. When the explants were stored 10 d after the subculture, higher percentages of survival and multiplication rates were obtained. The 4 species studied may be maintained at 2°C for up to 1 year without subculturing., The work was partially supported by the EEC (ECLAIR, AGRE-0067).

The effects of short-to medium-term cold storage (3-4°C) on the maintenance of embryogenic capacity and the germination and conversion of somatic embryo cultures of Camellia were investigated. Four culture lines maintained by repetitive embryogenesis were used: three C. japonica lines and one C. reticulata cv. Mouchang line. Increasing cold storage time (3, 6, 9, and 12 months), decreased the frequency of surviving embryo clusters and the mean number of surviving embryos per cluster. Eight week old embryogenic cultures were stored for one or two months, after which secondary embryos were isolated from the parent embryos and transferred to the germination medium. Two months of chilling significantly improved plantlet conversion abilities of secondary embryos, with rates ranging from 76–100% (depending on the embryogenic line) against 32–65%, respectively, in the absence of cold treatment. The incidence of secondary embryogenesis during germination also decreased after cold treatment. Cold storage of somatic embryos of Camellia reduced their competence for secondary embryogenesis while it increased their capacity for conversion to plantlets. These effects appear to reflect a general cold-induced increase in the maturity of the embryos.

Camellia (Theales, Theaceae) is a genus of tropical or subtropical trees and shrubs native to Eastern Asia. Its antiquity has been confirmed by fossils belonging to the Cretaceous period of the Secondary Era. The genus name was created by Linnaeus in the honour of the Jesuit missionary G.J. Kamel,who first recommended to grow the “Japanese rose” in Europe. In his Systerna Naturae of 1735, Linnaeus gave the name Camellia tsubaki to the plant indegenous to Japan as the tsubaki and it only acquired its present systematic name, Camellia japonica, in his Species Plantarum, in which he also described the tea plant, C. sinensis, under the name Thea sinensis. The systematics of the genus have since varied as the number of known members has increased; in particular, Chang and Bartolomew (1984) have carried out a major revision, grouping some 200 recognized species in four subgenera and twenty sections.

The aim of this study was to micropropagate mature Quercus robur L. trees when material retaining physiologically juvenile characteristics (stump sprouts, epicormic shoots) is not available. Branch segments from 70–300 year-old trees were force-flushed and the flushed, partially rejuvenated or reinvigorated shoots were used as a source of explants for establishment of cultures. In vitro establishment and multiplication was achieved with seven of the eight selected trees. The proliferation capacity of cultures of vertically placed explants declined after several subcultures, but efficient shoot multiplication was achieved by culturing decapitated shoots placed horizontally on GD medium supplemented with 0.89 μM of 6-benzyladenine. Reculturing the same horizontal explant several times allowed both higher multiplication rates and a shorter subculture cycle (2 weeks). An initial dark period of 5 days generally improved rooting capacity, which ranged, depending on clone, from 15 to 46%.

Somatic embryogenesis and plantlet regeneration were achieved from immature and mature zygoticCamellia japonica embryos cultured on Murashige & Skoog's mineral medium without growth regulators or with various combinations of IBA and BAR The dependence of embryogenesis rates on growth regulator levels was not clear, though high concentrations such as 4 mg 1-1BAP plus 2 mg 1-1IBA were definitely inhibitory. BAP at 1 or 2 mg 1-1 did appear to determine the formation of ‘bud-like embryos’. By far the most responsive initial explants were immature embryonic axes collected in September, 94% of which produced somatic embryos as against only 20% for embryonic axes from mature seeds collected in October. Cotyledon explants were also embryogenic. Somatic embryos differentiating directly on the hypocotyl of the embryonic axes or the surface of cotyledons passed through typical stages of embryogenesis. Indirect somatic embryogenesis via callus was also evident. Embryogenic potential was maintained by secondary embryogenesis through the successive generations of embryos.

SummaryIn order to obtain optimum conditions for in vitro propagation of Camellia japonica in its juvenile growth phase, several rooting experiments were performed. Dipping the basal ends of shoots in concentrated IBA solutions (0.5 and 1 g l−1) for short periods gave much better results than including the auxin in the rooting medium. Roots induced by IBA were better formed than when NAA was used. In general, the 1 g 1−1 IBA level gave better results than 0.5 g l−1 as regards both rooting rates and the number of roots per rooted shoot. Dipping times of 20–30 min were suitable although pronounced clonal differences were observed. In experiments on the sucrose concentration in the rooting medium, the best results were obtained using 30–50 g l−1, depending on the clone. Both rooting rates and the speed of root formation increased when cultures were incubated in the dark for 9–18 days prior to illuminated incubation. This fact must be related to the root initiation phase since the differentiation of root prim...

SummaryShoot cultures of Camellia japonica cv. Alba Plena have been established and multiplied in vitro using original material from a mature tree 50 years old. Three kinds of stage II expiants were distinguished for the purposes of shoot multiplication: ST1, the shoot tips of harvested shoots longer than 13–14 mm; n, nodal segments constituting the lower parts of the shoots from which the ST1 were taken; and ST2, whole harvested shoots 5–10 mm long. The ST1 explants were less responsive than the others. Of the various macro-nutrient formulae tried in shoot multiplication cultures the most satisfactory was Woody Plant Medium. The most suitable growth regulator combination for achieving shoot multiplication was: 2 mg l−1 of BA, 2 mg l−1 of zeatin, 2 mg l−1 of 2iP and 0.01 mg l−1 of IBA, used during four weeks, then the cultures were transferred to the same medium for another four weeks the cytokinin concentration being reduced to 1 mg l−1 (transfer medium). The addition of a liquid medium of the same compo...

Shoot cultures of Quercus robur have been established and multiplied in vitro using original material from both juvenile seedlings and mature trees. The juvenile explants were embryos and shoot apices from 3–4-month-old seedlings. Shoot tips from the new growth of stump sprouts were used to establish initial cultures of mature trees. Of the various macronutrient formulae tried, the most satisfactory were Heller’s medium with 1 mM of (NH4)2SO4, and Gresshoff and Doy’s. A dose of 6-benzylaminopurine at 1 mg l−1 was used in initial cultures and at 0.1 mg l−1 in shoot multiplication cultures. A rooting rate of 83% was achieved with juvenile material and 63% with adult material by briefly dipping the basal ends of shoots regenerated in vitro in concentrated solutions of IBA.

Alcohol coniferílico fue identificado de los exudados producidos por callos deCastanea sativa Mill. cultivados in vitro, originados de cotiledón.

Shoot-tip necrosis occurs during the rooting of chestnut and oak cultures. In some cultures, the role of apical dominance is taken over by one of the lateral buds, generally that nearest the apex, and the plant survives. Attempts were made to prevent necrosis by adding a low concentration of 6-benzyladenine (BA) to the rooting medium and, whilst necrosis was to a large extent avoided, the rooting rates fell considerably. Excision of the shoot-tip and application to the cut end of a drop of 25 mg l−1 BA in agar induced axillary growth. The optimum time for carrying out decapitation and BA treatment was after 10 days in the rooting medium. It is suggested that the cause of shoot-tip necrosis is the absence of cytokinin from the rooting medium linked with the root-inducing auxin treatment.

The potential benefits of establishing forests with selected vegetatively propagated clonal planting stock have long been recognized. Japanese records show that cuttings of Cryptomeria japonica have been planted continuously since around 1400 (Toda 1974). In the past decade, the potential for application of in vitro techniques to vegetative multiplication of forest trees has attracted a great deal of attention (Bonga 1974, 1977, Durzan 1980a, b, Dormling et al. 1976, Farnum et al. 1983, McKeand 1981, Mott 1982, Mott et al. 1977, Sommer and Brown 1979, Teo and Huhtinen 1975). The importance of tissue culture in forestry has recently been emphasized (Bonga and Durzan 1982).

Establishment of callus tissue culture from cotyledon fragments of Castanea sativa Mill. is described. The explants were grown on various combinations of auxins (IAA, IBA, NAA, 2,4-D) and cytokinins (K, BA) to induce callus and regeneration of organs. The best growth occurred on basal medium with 2,4-D (1 and 10 mg/l) plus both K or BA (0.5 mg/l). Root regeneration was achieved with both IBA and NAA (10 mg/l) supplemented with K or BA.

Starch depletion during rooting of juvenile chestnut cuttings was examined. A marked depletion occurred in the first 3 days following hormonal treatment; in untreated control cuttings there was no significant effect during the same period of time. Anatomical changes during the rooting-process were also studied. Greater starch depletion following hormonal treatment could provide the energy necessary for the great cell proliferation observed at 3–5 days, the organization of the primordia and the differentiation of the new xylem.