Foreed 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 M 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%.Abbreviations BA 6-benzyladenine - GD Gresshoff and Doy Medium - IBA indole-3-butyric acid     

Shoot elongation,leaf demography and bud formation in relation to branch position on Larix laricina saplings

Summary Shoot development was investigated on branches of Larix laricina (Du Roi) K. Koch trees growing in their 8th year in two plantations and in a natural stand approximately 12 years old. Expansion of throughout-crown series of short and long shoots was measured weekly, and later colour change and natural fall of leaves were assessed. Similar shoots were collected at intervals and dissected, the long shoots by 25-leaf segments. Leaf area and weight, as well as time of bud formation, were determined. Increasing acropetal trends were evident in time to bud burst: duration of short-shoot leaf-cluster expansion; size of leaf clusters and number, area and weight of leaves per cluster; duration and rate of long-shoot elongation; number, area and weight of leaves on long shoots; time to terminal-bud formation on long shoots. Along each long shoot, stem and leaf elongation and lateral-axis formation progressed acropetally. Lateral axes were most numerous on second to fourth 25-leaf segments. On longer shoots, some axes in middle segments developed as sylleptic short shoots rather than as lateral buds. Leaves of short shoots and basal leaves on long shoots turned yellow and abscissed sooner than axial leaves on long shoots. Colour change and loss among axial leaves were acropetal along shoots and up the crown. Thus, last-formed leaves, in axils of some of which lastformed lateral buds occurred, were held longest.     

A protocol for Ulmus minor Mill. micropropagation and acclimatization

Here we report the establishment of a simple protocol for the micropropagation and acclimatization of U. minor. Branches with dormant buds were collected from mature elms and sprouted in a greenhouse. Tip and node segments were used as starting material for in vitro proliferation in a medium (designated here as DKW1) already used for the micropropagation of a clone of the English Elm (U. procera SR4). In the first assay, in which explants from nine different trees were used, 88.5% of the tip segments produced new axillary shoots thus proving to be the best explant type. Afterwards, material from four different trees (F4, F7, F13, F14), that had the highest sprouting rate in the greenhouse, was used to test for genotype influence. F14 proved to be the best genotype in culture and it was used for all the subsequent experiments. Shoots from F14 were used to assay in vitro rooting using five DKW based media. Rooting percentages were high for all media and varied between 80% and 100%. For acclimatization two approaches were assayed: the use of previously rooted in vitro plants and the direct acclimatization of shoots from cultures in DKW1. After 6 weeks, 86.4% of the in vitro rooted plants were successfully acclimatized and a slightly higher value, 88.6%, was attained by direct acclimatization of shoots with thick stems and hard leaves. These results proved that there is no need for a previous in vitro rooting step and that direct acclimatization can effectively reduce time and costs. Thus U. minor micropropagation and acclimatization can be divided into only two steps: proliferation of shoots in DKW1 and direct acclimatization of these shoots in a sterile soil mixture.     

Basal reiteration improves the hydraulic functional status of mature Cinnamomum camphora trees

We compared soil-to-leaf hydraulic conductance (G _T), hydraulic conductivity and water-relations characteristics of leaves between reiterated axes (produced by sprouting of suppressed buds) and sequential axes (produced by elongation of terminal buds) on the same branch to investigate how basal reiteration affected the hydraulic architecture of mature Cinnamomum camphora (L.) Sieb. trees. Given similar light conditions, G _T was higher for leaves on reiterated shoots than for those on sequential shoots. However, where leaves on sequential shoots received more light, G _T was similar to that of leaves on reiterated shoots, suggesting that some compensatory mechanism worked to increase hydraulic conductance to the more distal sequential shoots, which have higher potential for carbon gain. Both xylem- and leaf-specific conductivities were higher for reiterated than sequential shoots. Pressure–volume measurements indicated that leaves on reiterated shoots were more vulnerable to water stress, suggesting that they developed under favorable water status. Because basal reiteration occurs on lower-order branch axes, reiterated shoots have better connectivity to higher conducting xylem and this may contribute to favorable water status. As trees grow larger, hydraulic pathlength and hydraulic resistance both increase as numbers of branch junctions and nodes increase. Our results suggest that basal reiteration improves the hydraulic functional status of mature C. camphora trees by shortening the hydraulic pathway and increasing hydraulic conductance to transpiring leaves.     

Micropropagation of juvenile and adult Sorbus domestica L.

Successful propagation of seedlings and mature trees of Sorbus domestica L. has been achieved by in vitro methods. Multiple shoot formation was obtained by placing shoot apices or nodal segments on a modified Schenck and Hildebrandt medium containing benzyladenine. Regenerated shoots were excised and induced to root on media with auxin. In the best treatments 75–85% of shoots from juvenile material rooted. Rooting capacity of shoots from mature explants was lower (30%) and was not improved by dipping the base of shoots in concentration solutions of indolebutyric or naphthaleneacetic acids. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid     

Plant regeneration through somatic embryogenesis from tissues of mature oak trees: true-to-type conformity of plantlets by RAPD analysis

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.     

Somatic Embryogenesis in Mature Quercus Robur Trees

Somatic embryo induction and plant regeneration have been obtained in tissues from mature Quercus robur L. trees. Epicormic shoots were forced to flush in branch segments collected from the crown of trees growing in selected stands on different collection dates. Expanding leaves from five genotypes, cultured following a multistage treatment procedure, produced somatic embryos at frequencies ranging from 0.3 to 3.6% of leaf explants, depending on genotype and collection date. After being induced, somatic embryos started a recurrent process by secondary embryogenesis which amplified the 15 embryogenic lines established. Plant recovery was achieved in 60% and 17% of matured embryos from two genotypes.     

Micropropagation of Ulmus pumila L. from mature trees

Summary Multiple shoots were induced from nodal segments of mature trees of Ulmus pumila L. on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA). Further multiple shoots were obtained from nodal segments taken from in vitro proliferated shoots when cultured in MS medium containing 0.5 mg.l^–1 BA. Rooting of the shoots was achieved on half or full strength MS medium or in MS medium supplemented with 0.1 mg.l^–1 naphtaleneacetic acid (NAA). Rooted plantlets were able to resume independent growth after a short period of acclimatization.     

In vitro propagation of birch (Betula verrucosa Ehrh)

Shoot t ps, young internodal segments and young developing leaves ofBetula ver rucosa Ehrh. in contact with agar nutrient medium formed tissue with numerous buds if medium contained a low concentration of cytokinin (BAP) and auxin (IBA or NAA). Tissue with induced buds transferred on a fresh nutrient medium continued in a formation of new buds which developed into shoots. Excised shoots were rooted on agar medium with a low concentration of auxin. Regenerated trees showed a genetic uniformity.     

Relationship between reproductive behavior and new shoot development in 5-year-old branches of olive trees (Olea europaea L.)

The development of new shoots plays a central role in the complex interactions determining vegetative and reproductive growth in woody plants. To explore this role we evaluated the new shoots in the olive tree, Olea europaea L., and the effect of fruiting on new shoot growth and subsequent flowering. Five-year-old branches served as canopy subunits in order to obtain a global, whole-tree view of new shoot number, size and morphological origin. The non-bearing trees had many more shoots than the fruit-bearing trees, and a greater number of longer shoots. In both bearing conditions, however, the majority of shoots were less than 4 cm long, with shoots of progressively longer lengths present in successively decreasing frequencies. Six major shoot types were defined on the basis of apical or lateral bud origin and of parent shoot age. On fruit-bearing trees, the new shoots originated predominantly from the shoot apex, while on non-fruiting trees, they formed mainly from axillary buds, but in both cases, they tended to develop on younger parent shoots. The previous bearing condition of the tree was the main determinant for subsequent inflorescence development, which was independent of both shoot type and length. Thus, reproductive behavior strongly affected both the amount and type of new branching, but subsequent flowering level was more influenced by previous bearing than by the potential flowering sites on new shoots.     

Impact of a gall-inducing aphid on Pistacia atlantica Desf. trees

The impact of gall-inducing aphids on shoot development was analyzed in 900 shoots from 20 pistachio trees, Pistacia atlantica Desf. (Anacardiaceae): 600 in which the axillary—lateral buds were galled by Slavum wertheimae HRL during the previous growth season, and 300 ungalled shoots. Although P. atlantica is a compensating tree, and the aphids do not attack the apical buds, further development of shoots from the apical buds was stopped in 62% of the galled shoots, while only 8.7% of nongalled shoots stopped their growth. Further development was stopped more often on shoots carrying two or more galls than on shoots supporting only one gall. To assess the hypothesis that bud destruction by the aphids explains this pattern, a field experiment was conducted in 140 shoots, distributed across seven trees. One, two or three axillary buds from five shoots of each tree were removed for each treatment, and five other shoots were marked as controls. Only 14 shoots (10%) of the 140 did not develop. The growth of the other shoots was not very different among the treatments. The colonization of the apical shoots, which developed on previously treated shoots, by three other galling aphid species was monitored. Removing lateral buds considerably reduced the establishment of Geoica sp. galls (70% of them colonized control shoots), but weakly influenced Forda riccobonii (Stefani). It also contributed only 5% of the total variance of the distribution of Smynthurodes betae West. The different results of the survey and the experiment show that the impact of S. wertheimae galls on the future growth of shoots from apical buds is more complex than the simple physical destruction of the axillary buds. Handling editor: Graham Stone     

In vitro clonal propagation of Lagerstroemia flos-reginae Retz

Multiple shoots were obtained from nodal segments of young and mature trees of Lagerstroemia flos-reginae Retz on MS medium with 7.50–20 mg/l of benzyl amino purine. Rooting was achieved on transfer of the excised shoots to MS medium with 1 mg/l of indole butyric acid. The plantlets have been successfully transferred to soil.     

Predation on Mindarus abietinus infestingbalsam fir grown as Christmas trees: the impact ofcoccinellid larval predation with emphasis on Anatis mali

The impact of natural coccinellid larvalpredation on the balsam twig aphid was evaluated bysystematically removing coccinellid egg masses in a6–8 year-old balsam fir (Abies balsamea)Christmas tree plantation in southwesternQuebec. Among coccinellid species hunting on firfoliage during development of Mindarus abietinusfundatrices in May, the indigenous Anatis mali was by far the most abundant and themain one to oviposit on trees. Comparison of trees onwhich coccinellid larval predation was excluded withcontrol trees showed that A. mali had a markedimpact both during and after the phase of rapid M. abietinus population growth that followedfundatrix maturation. On trees where coccinellidlarvae were allowed, aphid colonies became inactive(i.e. no live aphids in the colony) about two weeksearlier than on controls. A strong dampening effect onaphid density was also observed in those colonies thatremained active until the end of the aphid life cycle.Predation on aphid colonies reduced sexualsproduction, as the density of M. abietinusoverwintering eggs per shoot subsequently was reducedby 32%. Predation by coccinellid larvae occurred toolate to prevent needle damage to current year shoots,which affects the aesthetic value of Christmas trees.However, current year shoots measured in the mid-crownof trees late in the season were 19% longer on treeswhere aphid predation by coccinellid larvae wasallowed, compared with trees where they were excluded.Rearing all larval stages of A. mali on 4thinstar and adult sexuparae of M. abietinusindicated an average consumption of 269 aphids tocomplete larval development and pupate, which wasequivalent to at least seven colonies of M.abietinus at maximum aphid density at theexperimental site. Anatis mali is an importantnatural control factor of balsam twig aphid inChristmas tree plantations, hence its activity shouldbe protected and possibly stimulated by favourablepest management practices.     

In vitro plantlet regeneration of<Emphasis Type="Italic"> Schinopsis balansae</Emphasis> (Anacardiaceae)

A protocol for the in vitro regeneration of rooted plants from nodal single bud segments of 10-year-old Schinopsis balansae trees was developed. Nodal segments were harvested from actively growing shoots of plants grown from seeds and maintained in pots under greenhouse conditions, and from epicormic shoots obtained by forced flushing of branches. Culture of nodal segments on nutrient medium containing the mineral salts and vitamins of Murashige and Skoog medium at 1/4 strength (1/4 MS), supplemented with 100 mg l^–1 ascorbic acid, 3% sucrose, and 5–15 M 6-benzyladenine resulted in regeneration of multiple shoots. Rooting of regenerated shoots was observed in 1/4 MS medium with vermiculite as the substrate and supplemented with 7.5 M indolbutyric acid.     

Factors affecting in vitro clonal propagation of Prosopis cineraria

Genotype, age of tree, nature of explant and size (length and diameter), season of explant collection, explant position on medium, plant growth regulators and certain additives (ascorbic and citric acids, adenine sulphate, L-arginine, glutamine and ammonium citrate), incubation conditions, and subculturing period greatly influenced the in vitro clonal propagation of P. cineraria. The maximum number of 10–12 shoots were induced from the nodal shoot segment from pruned thorny adult trees on Murashige and Skoog's (MS) medium containing 0.1 mgl^-1 indole- 3-acetic acid (IAA)+2.5 mgl^-1 benzylaminopurine (BAP)+additives. Higher temperature (31+-2°C) and mixed (fluorescent and incandescent) light of 50 mol m^-2 s^-1 photon flux density for 12 h per day photoperiod favoured shoot induction and subsequent growth. Explants from thornless trees produced 6–8 shoots per explant on MS medium containing 0.1 mgl^-1 IAA+5.0 mgl^-1 BAP + additives. Nodal shoot segments obtained from root and stump sprouts produced multiple shoots. Root segments differentiated into multiple shoots on MS medium containing 0.5 mgl^-1 indolebutyric acid (IBA)+2.5 mgl^-1 BAP.Differentiated shoots multiplied best on MS medium containing 0.1 mgl^-1 naphthalene acetic acid (NAA)+1.0 mgl^-1 BAP + additives. To yield multiple shoots the original explant was transferred 6 times on fresh medium after harvesting the differentiated shoots. Shoots were rooted by pulsing with 100 mgl^-1 IBA for 4 h and then culturing on hormone-free half strength MS medium. Initial dark incubation for 5 days at high temperature (33±2°C) was found essential for root induction from shoots which was 63% within two weeks. The rooted plantlets contained a consistent number of chromosomes (2n=28). It is suggested that the protocol developed could be useful for cloning of mature and tested trees of P. cineraria.     

ANOMALOUS SECONDARY GROWTH IN LIANAS OF THE BIGNONIACEAE IS CORRELATED WITH THE VASCULAR PATTERN

Vascular pattern and anomalous secondary growth were studied in shoots of Clytostoma callistegoides, a liana having two types of phyllotaxy, one decussate and the other whorled. In shoots with decussate phyllotaxy, typical of bignoniaceous lianas, the vascular pattern has four major vascular strands that extend continuously from internode to internode, whereas in shoots having a whorled phyllotaxy the pattern has six major vascular strands. The first unidirectional cambium segments which result in the anomalous secondary growth were initiated precisely opposite each of the major vascular strands in both types of shoots. It is concluded that positioning of unidirectional cambium segments responsible for anomalous growth is correlated morphogenetically with the vascular pattern.     

In vitro plant regeneration from seedling explants of Stereospermum personatum D.C.: a medicinal tree

Padar (Stereospermum personatum, family Bignoniaceae) is a well-known medicinal tree. Its complete regeneration occurred through shoot bud culture in vitro. The seeds germinated sequentially on plastic trays and polyethylene bags for 21 days served as explants source. Nodal segments from the seedlings were established on MS medium supplemented with 4.44 μM BA, in which 86.6% nodes showed shoot bud elongation. Then, nodal segments from the developed shoots were cultured on MS medium with several BA concentrations; best shoot multiplication was obtained with 0.44 μM BA. In a second experiment where PVP was added to proliferation medium, nodal segments from developed shoots produced maximum 2.78 shoots per node. The nodal segments showed shoot multiplication up to seventh subculture on. Finally, shoots were rooted on MS medium with 2.46 μM IBA. The plants transferred to net pots containing coco-peat were acclimatized in green house, where more than 80% plants survived and grew normally.     

Acorn production is linked to secondary growth but not to declining carbohydrate concentrations in current-year shoots of two oak species

In trees, reproduction constitutes an important resource investment which may compete with growth for resources. However, detailed analyses on how growth and fruit production interact at the shoot level are scarce. Primary canopy growth depends on the development of current-year shoots and their secondary growth might also influence the number and size of fruits supported by them. We hypothesise that an enhanced thickening of current-year shoots is linked positively to acorn production in oaks. We analysed the effect of acorn production on shoot growth of two co-occurring Mediterranean oak species with contrasting leaf habit (Quercus ilex, Quercus faginea). Length and cross-sectional area of current-year shoots, apical bud mass, number of leaves and acorns, xylem and conductive area, number of vessels of acorn-bearing and non-bearing shoots were measured in summer and autumn. Nitrogen and carbohydrates analyses were also performed in stems and leaves of both shoot types. Stem cross-sectional area increased in acorn-bearing shoots when compared with non-bearing shoots for both species and such surplus secondary growth was observed since summer. In bearing shoots, the total transversal area occupied by vessels decreased significantly from basal to apical positions along the stem as did the xylem area and the number of vessels. Leaves of bearing shoots showed lower nitrogen concentration than those of non-bearing shoots. Carbohydrate concentrations did not differ in stems and leaves as a function of the presence of acorns. Such results suggest that carbohydrates may preferentially be allocated towards reproductive shoots, possibly through enhanced secondary growth, satisfying all their carbon demands for growth and reproduction. Our findings indicate that acorn production in the two studied oaks depends on shoot secondary growth.     

STRUCTURAL INVESTIGATIONS OF ASEXUAL REPRODUCTION IN NEPHROLEPIS EXALTATA AND PLATYCERIUM BIFURCATUM

Nephrolepis exaltata cv. Bostoniensis, the Boston fern, exhibits extreme stem dimorphism. The plant has orthotropic, dictyostelic shoots which bear pinnatifid leaves and plagiotropic, protostelic stolons which are aphyllous. Vegetative reproduction occurs by budding from primary and secondary stolons. Secondary stolons arise exogenously from derivatives of the apical cell of the primary stolon, whereas root primordia develop endogenously. Shoots develop in vivo when a creeping stolon makes contact with the substrate via extensive root proliferation. When stolon segments are excised and grown in vitro, secondary stolon primordia expand and initiate leaf primordia, forming new leafy shoots. In Platycerium bifurcatum, the staghorn fern, asexual propagation occurs on ageotropic roots ramifying among the basal nest fronds. Root bud initiation is marked by root tip hypertrophy following cortical parenchyma expansion. Root apical cell derivatives produce the bud apex; the root apical cell remains separate from the developing root bud. Superficially, vegetative reproduction in Nephrolepis and Platycerium appears to involve unusual organs. However, both ferns exhibit leafy bud development from distinct sites of origin, not from undetermined primordia or from direct transformation of root to shoot. Thus, distinctness of organ types is maintained in these two ferns and no evidence for interconvertibility of organ types has been found.     

Establishment of juvenile-like shoot cultures and plantlets from 4–16 year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) trees

A method was developed for the establishment of shoot cultures from Douglas-fir trees selected for outstanding growth and form in a 12-year-old genetic test. Vegetative buds from the lower crown were sterilized and grafted in vitro onto juvenile clonal rootstock. The rootstocks were produced from adventitious buds induced on cotyledons, and were maintained through micropropagation. Buds that established grafts slowly elongated into shoots, which were harvested and multiplied through micropropagation. Grafts often grew several new shoots which in turn could be harvested. In 1987, 2830 buds were grafted from 18 superior trees. Twenty nine grafts (1%) produced shoots which established 11 of the 18 trees in culture. Their appearance and behavior in vitro became more juvenile over 1–3 years, as indicated by shoot and needle morphology, disappearance of episodic growth pattern, increase in multiplication rates, and ability of needles to produce adventitious buds.The five most prolific of the 11 clones were given a pre-rooting treatment and planted in soil under fog. The success of rooting and subsequent establishment in soil varied from 5 to 17% depending on clone. In contrast, trees multiplied in vitro for 1–2 years longer showed soil establishment rates from 8–60%. This technique allows establishment, multiplication, and maintenance in vitro of cultures from high value Douglas-fir genotypes. Such cultures may serve as a starting point for further research on rejuvenation and cloning.