High frequency <Emphasis Type="Italic">in vitro</Emphasis> propagation of <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> from nodal buds of mature tree

An in vitro method for propagation of Holarrhena antidysenterica Wall. has been developed using nodal explants from mature trees growing in the field. Irrespective of concentrations and combinations of growth regulators used, the axillary and terminal buds sprouted and elongated when inoculated on Murashige and Skoog (MS) medium. The highest numbers of shoots were formed when sprouted shoots were subcultured from MS basal medium onto MS medium containing 2 mg dm⁻³ N⁶-benzyladenine (BA) and 0.5 mg dm⁻³ α-naphthalene acetic acid (NAA). The shoot number further increased upon subculture on MS medium containing 0.5 mg dm⁻³ BA. By repeated sub-culturing of shoots derived from nodal axillary buds, a high frequency multiplication rate was established. The elongated shoots were excised and rooted in auxin free MS basal medium. Ex vitro rooting of in vitro formed shoots was achieved upon dipping the microshoots for 2 min in 2 mg dm⁻³ of indole-3-butyric acid solution. Successful field establishment and high (80–90 %) survival of plants was observed.     

Variation in shoot mortality within crowns of severely defoliated <Emphasis Type="Italic">Betula maximowicziana</Emphasis> trees in Hokkaido,northern Japan

To clarify mortality patterns of current-year shoots within the crown of Betula maximowicziana Regel after severe insect herbivory in central Hokkaido, northern Japan, we investigated the degree of defoliation, pattern of shoot development, shoot mortality, and leaf tissue-water relations. One hundred current-year long shoots growing in a B. maximowicziana plantation were observed for defoliation and mortality in June 2002. An outbreak of herbivorous insects (Caligula japonica and Lymantria dispar praeterea) occurred in the stand in mid-to-late June, and the monitored shoots were defoliated to various degrees. Within 1 month of defoliation, some of the severely defoliated shoots had produced new leaves on short shoots that had emerged from axillary buds. Stepwise logistic regression revealed that the probability that current-year long shoots would put out axillary short shoots with leaves is closely related to the degree of defoliation. To evaluate the water relations of the leaves, we determined pressure–volume curves for the leaves that survived the herbivorous insect outbreak and the new leaves that emerged after defoliation. The water potential at turgor loss (Ψ_l,tlp) and the osmotic potential at full turgidity (Ψ_π,sat) were higher for the new leaves than for the surviving leaves, indicating a lower ability to maintain leaf cell turgor against leaf dehydration in the new leaves. Of the 100 shoots, 13 died after the emergence of new leaves. Stepwise logistic regression revealed that the probability that the long shoots would die generally increased with the emergence of new leaves, with increasing shoot height. This result suggests that the combined effect of the vulnerability of newly emerged leaves and low water availability, associated with higher shoot positions within the crown, caused shoot mortality. Based on our results, some possible mechanisms for mortality in severely defoliated B. maximowicziana are discussed.     

Plantlet regeneration from fascicular buds of seedling shoot apices of <Emphasis Type="Italic">Pinus roxburghii</Emphasis> Sarg

Shoot apices of Pinus roxburghii Sarg were cultured on Murashige and Skoog’s medium (MS) supplemented with cytokinins [6-benzyladenine (BA), kinetin and N-benzyl-9-(2-tetrahydropyranyl) adenine (BPA)] alone and in combination with auxin, α-napthaleneacetic acid (NAA). Of the three cytokinins tested at varying concentrations, medium supplemented with 10 μM BA was found optimal in respect of explant responsiveness (97.22 %) and average number of buds induced per explant (7.42). The concentration of cytokinins in the induction medium had a profound effect on rate of elongation of induced buds on MS basal medium containing 0.5 % activated charcoal. Further, shoots induced on lower concentrations of BA increased up to 2.4 times in length in 4 weeks. Decapitation of the explant enhanced the rate of axillary bud elongation. Proliferating shoot cultures were established by sub-culturing the axillary shoots on MS supplemented with 10 μM BA. Shoots 2–3 cm in length were suitable for culturing as more buds were induced on them compared to longer or shorter shoots. Root primordia were induced on 70.83 % shoots when transferred to 1/2 MS medium supplemented with 5.0 μM NAA. Elongation of root primordia (60 %) was achieved in liquid 1/2 MS basal medium. The plantlets were successfully transferred to soil after hardening; the time period from initiation of shoot buds to transplantation being 20–22 weeks.     

Periods of organogenesis in mono- and bicyclic annual shoots of Juglans regia L. (Juglandaceae)

The organogenetic cycle of shoots on main branches of 4-year-old Juglans regia trees was studied. Mono- and bicyclic floriferous and vegetative annual shoots were analysed. Five parent annual shoot types were sampled between October 1992 and August 1993. Organogenesis of summer growth units was monitored between 16 Jun. and 3 Aug. 1993. Variations over time in the number of nodes, cataphylls and embryonic green leaves of terminal buds were studied. The number of nodes of parent shoot buds was compared with the number of nodes of shoots derived from parent shoot buds. The spring growth units of mono- and bicyclic shoots consist exclusively of preformed leaves which were differentiated, respectively, during the spring flush of growth (mid-April until mid-May) or the summer flush of growth (mid-June until early August) in the previous growing season. Thus, winter buds may consist of flower and leaf primordia differentiated in two different periods during annual shoot extension. The summer growth units of bicyclic shoots consist of preformed leaves that were differentiated in spring buds during the spring flush of growth in the current growing season. Bud morphology is compared between spring and summer shoots.     

The apical bud as a novel explant for high-frequency in vitro plantlet regeneration of <Emphasis Type="Italic">Perilla frutescens</Emphasis> L. Britton

In this study, we established an in vitro regeneration system to maximize the recovery of leafy perilla (Perilla frutescens L. Britton) plantlets as part of developing a molecular biotechnology-based metabolic engineering program for this crop plant. Hypocotyl segments including the apical buds were used as explants for the direct production of shoots without an interim callus phase. The number of shoots produced from the apical buds peaked within 3–4 weeks, and the shoots were subsequently cultured on Murashige and Skoog (MS) media supplemented with 2 mg l⁻¹ benzylaminopurine (BA). Spontaneous rhizogenesis was observed after 7–10 days of culture on MS media without hormonal additives. The rooted shoots developed into normal plants in soil after hardening on distilled water for 3–4 days. The average plantlet regeneration frequency was higher for the apical buds (64.33%) than for the top (15.66%), middle (4%), and basal (1.33%) segments of the hypocotyls. This regeneration system demonstrates a capacity for high-frequency plantlet recovery and thus should be considered for use in the genetic manipulation of leafy perilla.     

Development of shoot inHydrangea macrophylla I. Terminal and axillary buds

The structure of shoots, in particular of winter buds, ofHydrangea macrophylla was examined. The non-flower-bearing shoot is usually composed of a lower and an upper part, between which a boundary is discernible by means of a distinctly short internode. This internode is the lowermost of the upper part, and it is usually shorter than the internodes immediately above and below, although the internodes tend to shorten successively from the proximal to the distal part of the shoot. Variations exist in the following characters among the terminal bud, the axillary bud on the lower part of the shoot and the axillary bud on the upper part: (1) length of bud; (2) character of the outermost pair of leaf primordia; (3) degree of development of secondary buds in the winter bud; and (4) the number of leaf primordia. Usually, the terminal bud contains several pairs of foliage leaf primordia with a primordial inflorescence at the terminal of the bud, but the axiallary bud contains only the primordia of foliage leaves in addition to a pair of bud scales.     

Induction of adventitious buds in cultured petal explants ofChelidonium majus L.

Petal explants ofChelidonium majus L. (Papaveraceae) formed noteworthy adventitious buds without any intermediate callus when cultured under appropriate conditions. Bud formation was favored by combinations of 1–2 mg/l indoleacetic acid (IAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–0.5 mg/l kinetin (K). In the present study, neither bud formation nor callus formation occurred in cultures of excised leaves. A histological study revealed that adventitious bud formation occurred only in single epidermal layers of petals, while several subepidermal parenchyma layers did not join in its formation. Activation zones arising from the epidermis underwent intense cell divisions to initiate buds on the epidermal surface. These buds later turned green in color, developing into shoots which eventually grew into plantlets after root formation.     

Plant regeneration from excised hypocotyl explants of <Emphasis Type="Italic">Platanus acerifolia</Emphasis> willd

Summary A method of plant regeneration from hypocotyl segments of Platanus acerifolia Willd, has been developed. Hypocotyl slices were cultured on Murashige and Skoog (MS) basal medium supplemented with a range of combinations of cytokinins [6-benzyladenine (BA) or kinetin] and auxins [indole-3-butyric acid (IBA), indole-3-acetic acid, α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid] for adventitious shoot induetion. The highest regeneration frequency was obtained with MS medium containing 2.0 mg l⁻¹ (8.88 μM) BA and 0.5 mg l⁻¹ (2.46 μM) IBA. Adventitious buds and shoots were differentiated from hypocotyl-derived cellus or directly from the wounded sites within 4–8 wk. The regenerated shoots were elongated and proliferated efficiently on multiplication medium. Complete plantlets were transplanted to the soil and grew normally in the greenhouse after root formation on rooting medium for 4–6 wk.     

Size-dependent perennation bud formation and its effect on seedling establishment inVeronicastrum sibiricum ssp.japonicum

Seedling establishment of a perennial,Veronicastrum sibiricum ssp.japonicum colonizing at 1,600 m altitude in Nikko National Park was studied for two years. About half of the first-year seedlings died during the growing season and 45–74 per cent of the seedlings surviving the first summer died during the following winter season. This winter mortality depended on the end-of-season size of the seedling in the previous autumn and was caused mainly by the absence of perennation buds. Seedlings which had emerged on bare ground attained larger size and had higher winter survival than seedlings in a mat of moss. Seedlings germinating in early season attained larger size and had higher probability of winter survival than seedlings germinating in later season.     

Influence of phenylacetic acid on clonal propagation of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> wight &; ARN: An endangered shrub

Summary We have developed a highly efficient two-stage protocol for induction of multiple shoots from single node in vitro shoot tip explants of Decalepis hamiltonii. It was found that phenylacetic acid (PAA) had a synergistic effect on shoot multiplication when treated with N⁶-benzyladenine (BA). This protocol used PAA for both multiple shoot induction from nodal explants, elongation of primary shoots, and initiation of adventitious shoot formation from primary shoots. Murashige and Skoog medium containing BA (2.22–31.08 μM) and α-naphthaleneacetic acid (0.27–10.74 μM) or PAA (7.34–36.71 μM) was used to initiate shoot formation from nodal explants. The maximum number of shoots per culture was produced on a medium containing 31.08 μM BA and 14.68 μM PAA, while the longest shoot length and nodes were obtained on medium containing 22.2 μM BA and 14.68 μM PAA. Shoots subcultured on MS medium containing 22.2 μM BA and 14.68 μM PAA elongated along with secondary shoot formation. The shoots were rooted on medium containing 9.7 μM indole-3-butyric acid. The plantlets were acclimatized in soil with an 80–90% survival rate under field conditions.     

Developmental disorders in buds and needles of mature Norway spruce, Picea abies (L.) Karst., in relation to needle boron concentrations

Dieback of the terminal shoot and consequently bushy growth induced by boron deficiency have been reported widely throughout the world in several tree species. Recently, similar growth damage was documented in half of the young spruce stands in eastern Finland. To clarify the role of B deficiency, the light microscopic structure of emerging buds and of developing and previous-year needles of mature Norway spruce (Picea abies L. Karst.) from damaged (D stand), partly damaged (PD stand) and healthy (H stand) stands were analysed. The samples, on which needle nutrient concentrations were also determined, were taken seven times between early spring (April) and early winter (November). Cell death characterized by precipitation of the cell content, possibly due to the release of tannins after membrane rupture, was seen in the apex of emerging buds, and this led to fatal damage in about half of the buds in the trees from the D stand, where the needle B concentration was well below the deficiency level of 4–5 mg kg⁻¹. Furthermore, an increase in living cells that accumulated tannins in the vacuoles, which is a common stress and/or defense reaction, was found in the primordial shoots of buds and in the differentiating needles in the PD and D stands. The increase in the areas of the central cylinder and of the xylem found in the needles indicate structural plasticity during needle differentiation to drought. The time frame for bud emergence from late May up to mid-September means that an adequate B supply is necessary throughout the summer in order to avoid fatal bud damage and thus bushy growth of the trees.     

Regeneration of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.) from root explants

Summary Eggplant (Solanum melongena L.) was efficiently regenerated from cultured roots of 15-d-old seedlings on Murashige and Skoog (MS) medium containing 0.45 μM thidiazuron and 13.3 μM 6-benzyladenine. Within 28d of culture initiation, induction of organogenic calluses and subsequent differentiation into shoot buds were observed. Shoot buds upon subculture to MS basal medium elongated into healthy shoots. Excised shoots (2–4 cm) were rooted on Soilrite^? irrigated with water either in vitro or in vivo. Plants with well-developed root systems were established under field conditions after hardening in the glasshouse, where they developed into flowering plants and produced mature fruits with viable seeds.     

Resprouting after disturbance in the short-lived herb Rorippa palustris (Brassicaceae): an experiment with juveniles

The impact of plant age, severity of injury and nutrient levels on the ability to resprout from roots was experimentally assessed in juveniles of the short-lived herb Rorippa palustris (L.) Besser. In a chamber experiment, six cohorts of young plants (1–6 week old) were injured to obtain data on the threshold age for the ability to resprout from roots. We found that plant age was an important factor influencing resprouting ability: injured individuals older than 5 weeks were able to resprout, but not plants younger than 3 weeks. The impact of injury severity (defoliation and removal of axillary buds) and nutrient levels on resprouting ability was assessed on juveniles in a greenhouse experiment. Injury induced growth of new shoots from root buds, while the number of adventitious buds on roots was not influenced by injury. Both injury treatments had a similar effect in this respect, and the amount of regenerated biomass and the extent of regeneration were not different among injury treatments. The number of new shoots produced after injury was higher at the high nutrient level, but the number of formed adventitious buds on roots was not influenced by nutrient level. Nutrient level also influenced the amount of regenerated biomass, but the extent of regeneration (regenerated/removed biomass) was not influenced. The short-lived monocarpic species R. palustris is able to resprout from roots relatively easily. This ability seems to be advantageous in disturbed habitats and this idea is discussed throughout the paper.     

Clonal micropropagation of <Emphasis Type="Italic">Crataeva adansonii</Emphasis> (DC.) Prodr.: A multipurpose tree

Summary A method of micropropagation through multiple shoot formation from axillary buds of mature tree and rootstock growths of Crataeva adansonii (DC.) Prodr. (a multipurpose tree) has been developed. Factors affecting multiplication rate included season, age of explant source, explant type, type of bud, position of bud on the foliage twig, type of medium, various additives, and explant implantation on the medium. The maximum number of buds was produced from the sixth to 10th axillary buds taken from foliage twigs of 40–50-d-old rootstock growth in the months of October to December. At this time of the year the contamination was minimum. Optimum response was recorded on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 3 mgl⁻¹, 13.3μM) and 1-naphthaleneacetic acid (NAA; 0.05 mgl⁻¹, 0.27 μM) after 21 d of culture. Shoot buds were further multiplied and maintained on BA (1–1.5 mgl⁻¹, 4.4–6.7 μM) while shoots elongated on BA (0.1–0.5 mgl⁻¹, 0.44–2.2 μM) supplemented medium. The number of shoots was further multiplied by using nodal segments of in vitro-regenerated shoots as microcuttings and repeated subculturing of stumps after excising the microshoots. In vitro rooting on growth regulator-free MS medium was possible with 70% of microshoots after 4 wk. From one nodal segment 150 plantlets were produced within 14 wk.     

Long-term changes in tree-ring–climate relationships at Mt. Patscherkofel (Tyrol,Austria) since the mid-1980s

Although growth limitation of trees at Alpine and high-latitude timberlines by prevailing summer temperature is well established, the loss of thermal response of radial tree growth during last decades has repeatedly been addressed. We examined long-term variability of climate–growth relationships in ring width chronologies of Stone pine (Pinus cembra L.) by means of moving response functions (MRF). The study area is situated in the timberline ecotone (ca. 2,000–2,200 m a.s.l.) on Mt. Patscherkofel (Tyrol, Austria). Five site chronologies were developed within the ecotone with constant sample depth (≥19 trees) throughout most of the time period analysed. MRF calculated for the period 1866–1999 and 1901–1999 for ca. 200- and ca. 100-year-old stands, respectively, revealed that mean July temperature is the major and long-term stable driving force of Pinus cembra radial growth within the timberline ecotone. However, since the mid-1980s, radial growth in timberline and tree line chronologies strikingly diverges from the July temperature trend. This is probably a result of extreme climate events (e.g. low winter precipitation, late frost) and/or increasing drought stress on cambial activity. The latter assumption is supported by a <10% increase in annual increments of ca. 50-year-old trees at the timberline and at the tree line in 2003 compared with 2002, when extraordinary hot and dry conditions prevailed during summer. Furthermore, especially during the second half of the twentieth century, influence of climate variables on radial growth show abrupt fluctuations, which might also be a consequence of climate warming on tree physiology.     

Development of shoot inHydrangea macrophylla II. sequence and timing

The development of axillary buds, terminal buds, and the shoots extended from them was studied inHydrangea macrophylla. The upper and lower parts in a nonflower-bearing shoot are discernible; the preformed part of a shoot develops into the lower part and the neoformed part into the upper part (Zhou and Hare, 1988). These two part are formed by the different degrees of internode elongation at early and late phases during a growth season, respectively. Leaf pairs in the neoformed part of the shoot are initiated successively with a plastochron of 5–20 days after the bud burst in spring. The upper axillary buds are initiated at approximately the same intervals as those of leaf pairs, but 10–30 days later than their subtending leaves. Changes in numbers of leaf pairs and in lengths of successive axillary buds show a pattern similar to the changes in internode lengths of the shoot at the mature stage. The uppermost axillary buds of the flower-bearing shoot often begin extending into new lateral shoots when the flowering phase has ended. The secondary buds in terminal and lower axillary buds are initiated and developed in succession during the late phase of the growth season. Internode elongation seems to be important in determining the degrees of development of the axillary buds. Pattern of shoot elongation is suggested to be relatively primitive. Significances of apical dominance and environmental conditions to shoot development are discussed.     

Stimulation of the regeneration capacity of tree shoot segment explantsin vitro

Regeneration abilities of buds on shoot segment explants isolated from adult trees of oak (Quercus robur), aspen (Populus tremula), black locust (Robinia pseudacacia), Japan pagoda tree (Sophora japonica), and English walnut (Ailanthus glandulosa) were studied during the growing season. Optimum BAP concentrations for the regeneration of oak bud meristems were dependent on the date of sampling. Axillary shoots could be induced from winter and summer buds of oak and aspen on Dustan and Short media supplemented with activated charcoal and BAP at concentrations from 0.5 to 2 mg 1^-1. More intensive rooting of segments of newly formed shoots was observed on MS medium diluted to one half and supplemented with 2 % sucrose and 0.2 mg 1⁻¹ of IBA.Populus tremula formed longer axillary shoots on DS media supplemented with 0.5 mg 1⁻¹ of BAP and 1 mg 1⁻¹ of GA₃. Regeneration capacities of black locust, Japan pagoda tree, and English walnut were higher. In addition to the induction of multiple shoots from buds, shoots could also be obtained from calluses formed on basal segment parts. Asparagine and glutamine at a concentration of 25 mg 1⁻¹ stimulated the percentage of differentiated stems on callus surface. Inhibitory effects of substances which accumulated in buds in the second half of the growing season could be reduced by means of pulse treatments in 50 mg 1⁻¹ BAP solutions or using short-term dipping into 0.1 % AgNO₃ solution.     

Strawberry sepal: Another explant for thidiazuron-induced adventitious shoot regeneration

Summary An efficient system to regenerate shoots on excised sepals (calyx) of greenhouse-grown ‘Bounty’ strawberry (Fragaria x ananassa Duch.) was developed in vitro. Sepal cultures produced multiple buds and shoots without an intermediary callus phase on 2–4 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ)-containing shoot induction medium within 4–5 wk of culture initiation. Young expanding sepals with the adaxial side touching the culture medium and maintained for 14 d in darkness produced the best results. In a second experiment, sepals proved more effective than the leaf discs and petiole segments for regenerating shoots. A third experiment compared the effects of six concentrations of two cytokinins (TDZ at 0, 0.5, 2, and 4 μM and zeatin at 2 and 4 μM) for elongation of sepal-derived adventitious shoots. The media containing TDZ generally promoted more callus formation and suppressed shoot elongation. TDZ-initiated cultures transferred into the medium containing 2–4 μM zeatin, produced usable shoots after one additional subculture. Shoots were rooted in vitro in the same medium used for shoot regeneration, but without any growth regulators. When transferred to potting medium, 85–90% of in vitro plantlets survived.     

In vitro shoot-tip grafting improves recovery of cotton plants from culture

A rapid in vitro shoot-tip grafting (STG) technique was adapted to increase recovery of intact cotton plants from shoots developed in culture. Induction of root organogenesis in cotton shoots is genotype dependent and unreliable. The resulting loss of regeneration potential due to failure to form roots can vary from 30 to 80% according to genotype and represents a significant bottleneck in the overall recovery of plants from culture. If the non-rooting shoots are transgenic, the loss in regenerated plant material can be substantial. In vitro grafting of cotton shoots to seedling rootstock proved to be a simple and reliable method allowing 90–100% recovery of non-rooting shoots from culture. Success of any given graft was directly related to scion size (0.8–1.0 cm) and age (14–35 days) of the seedling rootstock. The method appeared to be genotype independent, and varietal differences between rootstock and scion did not effect the rate of plant recovery from culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Bud and shoot structure may relate to the distribution area of South American Proteaceae tree species

The capacity of preformation and neoformation and the structure of winter buds are vegetative attributes that may vary between plant species and according to ontogenetic stages of the same species. The present study describes and evaluates these features for the four tree species of Proteaceae occurring in Patagonia. In particular, it analyzes the structure and development of the distal buds of the trunk and the preformed or neoformed nature of the organs involved. Two of the species, Embothrium coccineum and Lomatia hirsuta, have scaly buds, in which primordia of green leaves are covered by cataphylls. The shoots of both species may include neoformed organs, more frequently so in juvenile trees. Lomatia ferruginea and Gevuina avellana have naked buds with a low number of primordia; in juvenile and adult trees of both species trunk shoots are entirely preformed. The structure of buds and shoots suggests two different growth modalities of the axes, which would be related to ecological breadth (narrower in the species with naked buds than in those with scaly buds) and distribution area of these species. The considerable morphological differences between the two Lomatia species studied raise the question whether they ought to be included in the same genus.