Ana M. Vieitez

In vitro plantlet regeneration from juvenile and mature Quercus robur, L.

SummaryShoot 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.

Cold storage of shoot cultures and alginate encapsulation of shoot tips of Camellia japonica L. and Camellia reticulata Lindley

Abstract 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 Skoogs 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.

Forced flushing of branch segments as a method for obtaining reactive explants of mature Quercus robur trees for micropropagation

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 plant regeneration from embryonic tissues ofCamellia japonica L.

Somatic embryogenesis and plantlet regeneration were achieved from immature and mature zygoticCamellia japonica embryos cultured on Murashige & Skoogs 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.

Culture of chestnut shoots from buds in vitro

SummaryA method is described of growing chestnut shoots in vitro from lateral buds of 3-4 month-old seedlings cultured in a nutrient medium. The addition of BAP to the culture medium, optimally at a concentration of 1 mg l-1 caused the development of axillary shoots. Zeatin induced more vigorous shoots, but with little development of axillary shoots. Kinetin had no effect, compared to the control, without cytokinin. An adverse effect on growth was observed when GA3 was added to the cultures. Sub-cultures of single shoots and nodes excised from shoots on the initial cultures were established in a medium with 0.1 mg l-1 BAP to promote further shoot multiplication.

Cryopreservation of Zygotic Embryonic Axes and Somatic Embryos of European Chestnut

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.

Improving micropropagation conditions for adult-phase shoots of chestnut

SummaryThis 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 m...

Application of biotechnological tools to Quercus improvement

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.

In vitro response of encapsulated somatic embryos of camellia

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.

Prevention of shoot-tip necrosis in shoot cultures of chestnut and oak

Vieitez, A.M., Sdnchez, C. and San-Jos~, C., 1989. Prevention of shoot-tip necrosis in shoot cultures of chestnut and oak. Scientia Hortic., 41: 151-159. 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 1-1 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 rootinducing auxin treatment.