Genotype Affects the Morphogenic Response in vitro of Epicotyl Segments of Citrus Rootstocks

The influence of light, hormones and explant orientation onin vitro regeneration in epicotyl cuttings was compared in fourCitrus species (C. aurantium, C. macrophylla, C. reshni andC.sinensis ) and the hybrid Troyer citrange (C. sinensis x Poncirustrifoliata). In all cases, explants planted vertically regeneratedshoots at the apical end by a process of direct organogenesiswithout callus formation. When the Troyer citrange explantswere incubated horizontally, regeneration at the apical endoccurred by an indirect organogenic pathway after callus formation.This change in the pathway of regeneration did not occur inany of the Citrus species, and incubation horizontally resultedin a reduction in the number of buds and shoots formed throughthe direct organogenic pathway. Shoot formation through thedirect organogenic pathway was inhibited by darkness, and thisinhibitory effect was counteracted by the cytokinin benzyladeninein Troyer citrange and, partly, in C. sinensis, but not in C.macrophylla. A non-organogenic callus formed at the basal endof most of the cuttings of C. reshni. InC. sinensis and C. aurantium,a non-organogenic callus formed only in a low proportion ofexplants. Troyer citrange formed an organogenic callus in whichbuds or roots differentiated depending on the auxin/cytokininbalance. C. macrophylla formed callus in the dark but not inthe light. Root formation occurred both in the presence of theauxin naphthaleneacetic acid or low concentrations (2.2 to 4.4µM) of the cytokinin benzyladenine, but no buds were formed.These qualitative and quantitative differences in the organogenicresponse indicate that the conditions for regeneration mustbe optimized for each genotype. Copyright 2000 Annals of BotanyCompany Benzyladenine, citrus, Citrus aurantium, Citrus macrophylla, Citrus sinensis, Citrus sinensis x Poncirus trifoliata, naphthaleneacetic acid, organogenesis, rooting, shoot regeneration, Troyer citrange     

Direct and Indirect Shoot Organogenic Pathways in Epicotyl Cuttings of Troyer Citrange Differ in Hormone Requirements and in their Response to Light

Bud differentiation by direct organogenesis at the apical endof Troyer citrange (Citrus sinensis[L]. OsbeckxPoncirus trifoliata[L].Raf.) epicotyl cuttings inserted vertically in a semi-solidculture medium did not require hormone additions. The numberof buds regenerated was slightly, but significantly, increasedwhen the incubation was performed in the light as compared tothe dark, and by the addition of benzyladenine (BA; 2.2 to 22µM) to the medium. Bud sprouting and subsequent shootformation required the addition of BA and was increased by lightto a higher extent than bud formation. The best response wasobtained with the highest BA concentration tested (22 µM).Regeneration through the indirect organogenic pathway at thetwo edges of the epicotyl cuttings when in contact with theculture medium did not occur in the absence of benzyladenine,which was an absolute requirement for callus development. Thebest regeneration response was obtained when the explants wereincubated in the light in the presence of 4.4 µM BA andan auxin. Indole-3-acetic acid (IAA; 5.8 µM) was moreeffective in increasing shoot formation than naphthaleneaceticacid (NAA; 0.54 µM). Higher NAA concentrations inhibitedshoot formation. Incubation in the dark or increasing the BAconcentration (22 µM) increased markedly callus growth,but inhibited both bud differentiation and sprouting, almostcompletely suppressing shoot formation. The conditions duringregeneration affected the rooting of the regenerated shoots.Rooting of 86% of the shoots was achieved in a medium with 2.7µM NAA and 2.6 µM indole-3-butyric acid. All therooted explants acclimated and survived transplanting. Underthe optimal conditions tested, the proliferation rate obtainedthrough the indirect regeneration pathway ranged from 60 to86 plants per seedling. Copyright 2000 Annals of Botany Company Troyer citrange, Citrus sinensisxPoncirus trifoliata, auxins, benzyladenine, direct organogenesis, hormone requirement, indirect organogenesis, light, morphogenesis, rooting.     

The influence of explant orientation and contact with the medium on the pathway of shoot regeneration in vitro in epicotyl cuttings of Troyer citrange

The effect of orientation as regards to gravity, and that of contact with the medium of culture, on shoot regeneration at the cut edges of epicotyl explants of Troyer citrange (Citrus sinensis (L.) Osbeck×Poncirus trifoliata (L.) Raf.) have been separated. The shoot regeneration pathway was not affected by the orientation of the explants as regards to gravity, and was determined by explant polarity and the contact with the culture medium. At the apical edge of the explants, the contact with the medium shifted the pathway of shoot regeneration from a direct one to an indirect one, with formation of a callus. This callus formation was cytokinin-dependent, but the change in the pathway of organogenesis was not caused by the increase in cytokinin availability resulting from the contact with the medium. In contact with the media, regeneration at the basal edge of the explants occurred through an indirect pathway after callus formation. No regeneration occurred, at the basal edge, if the contact with the media was prevented. The orientation of the explants as regards to gravity affected shoot formation through the direct pathway of organogenesis. The number of buds differentiated, and that of growing shoots increased when the orientation of the explants departed from the vertical upright position.     

Micropropagation of chinese redbud (<Emphasis Type="Italic">Cercis yunnanensis</Emphasis>) through axillary bud breaking and induction of adventitious shoots from leaf pieces

Summary Factors affecting in vitro shoot production and regeneration of Cercis yunnanensis Hu et Cheng were investigated by comparing various growth regulators and explant types. For optimum shoot production from axillary buds, Murashige and Skoog (MS) media containing 6-benzyladenine, either alone or in combination with a low concentration of thidiazuron, resulted in the greatest number of shoots formed per explant (>3). Explants (2 mm long) containing one axillary bud placed in directcontact with the medium yielded the most shoots per bud (1.6) when grown on growth regulator-free medium. Root formation on 70–80% of shoot explants was accomplished using either indole-3-butyric acid or α-naphthaleneacetic acid in the medium, with significantly more roots formed on explants possessing and apical bud than those without the bud. Direct shoot organogenesis from leaf explants occurred on MS medium containing 10–30 μM thidiazuron, with up to 42% of leaf explants producing shoots.     

Effect of phytoregulators and physiological characteristics of the explants on micropropagation of Maytenus ilicifolia

Micropropagated shoots of Maytenus ilicifolia Mart. were obtained from axillary buds cultured in Murashige & Skoog medium supplemented with 13.3 M 6-benzyladenine (BA). Addition of 1.1 M 1-indole-3-acetic acid (IAA) to the medium increased shoot elongation. The number of shoots formed was influenced by BA concentration, degree of juvenility of the explant, and by bud explant position on the stem. Cultures of buds taken from stem parts located close to the shoot tip yielded more callus than shoots, whereas axillary buds at distant positions from the apical bud yielded more shoots.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic-acid     

Bud Structure in Relation to Shoot Morphology and Position on the Vegetative Annual Shoots of Juglans regia L. (Juglandaceae)

The length and basal diameter of all lateral and terminal budsof vegetative annual shoots of 7-year-oldJuglans regia treeswere measured. All buds were dissected and numbers of cataphylls,embryonic leaves and leaf primordia were recorded. Each axillarybud was ranked according to the position of its associated leaffrom the apex to the base of its parent shoot. Bud size andcontent were analysed in relation to bud position and were comparedwith the size and number of leaves of shoots in equivalent positionswhich extended during the following growing season. Length andbasal diameter of axillary buds varied according to their positionon the parent shoot. Terminal buds contained more embryonicleaves than any axillary bud. The number of leaves was smallerfor apical and basal axillary buds than for buds in intermediatepositions on the parent shoot only. All new extended shootswere entirely preformed in the buds that gave rise to them.Lateral shoots were formed in the median part of the parentshoot. These lateral shoots derived from buds which were largerthan both apical and basal ones. Copyright 2001 Annals of BotanyCompany Juglans regia L., Persian walnut tree, branching pattern, preformation, bud content, shoot morphology     

Topophysis affects the Potential of Axillary Bud Growth, Fresh Biomass Accumulation and Specific Fresh Weight in Single-stem Roses (Rosa hybrida L.)

Topophysis, the effect on growth and differentiation of positionof axillary buds along the shoot, was studied by propagatingfive-leaflet-leaf single-node cuttings which were excised fromseven stem positions and grown as single stemmed plants. InRosahybrida ‘Korokis’ Kiss®, ‘Tanettahn’Manhattan Blue®, and ‘Sweet Promise’ Sonia®,following release of the buds from apical dominance by excision,morphogenetic development was studied until anthesis. The timefrom excision/planting until onset of bud growth, visible flowerbud appearance, and anthesis was generally shorter in plantsoriginating from apical bud positions than from basipetal positions.Topophysis mainly affected the onset of axillary bud growth;the earliest growth and development was found in cuttings fromthe second uppermost node position. This node tended to havethe lowest plastochron value, which indicated the existenceof a transition between sylleptic and proleptic buds. Stem lengthat visible flower bud and at anthesis generally increased asthe cutting position changed basipetally until the second lowestposition, and the number of five-leaflet-leaves at anthesisand the total number of nodes generally increased basipetally.For internode length, growth rate, and fresh biomass efficiencythe cuttings taken from the uppermost and lowermost positionsgenerally had significantly lower values than cuttings fromall medial positions. At anthesis, plants originating from cuttingsexcised from lower medial positions generally had a higher freshweight, greater flower stem diameter, and a significantly higherspecific fresh weight than those plants originating from apicalor basal positions. Among the cultivars, Sonia was the mostefficient in increasing fresh biomass and had the highest growthrate, whereas Manhattan Blue possessed the highest specificfresh weight, indicating a higher plant quality. It is suggestedthat topophysis inRosa is an independent phenomenon intrinsicto the axillary bud. apical dominance; axillary bud growth; fresh biomass accumulation; cut rose; flowering; Rosaceae; Rosa hybrida L.; rose; shoot growth; single-stem roses; specific fresh weight; topophysis; quality     

Role of polarity in de novo shoot bud initiation from stem disc explants of <Emphasis Type="Italic">Curculigo orchioides</Emphasis> Gaertn. and its encapsulation and storability

The occurrence of strong polarity towards shoot bud induction and the effect of cytokinin(s) on each segment of stem axis, encapsulation and storability of de novo Shoot buds of Curculigo orchioides Gaertn. (Hypoxidaceae) have been documented in the present communication. Maximum number of shoot buds arising de novo from the stem discs (cross section) explanted from proximal end on MS medium fortified with BAP and KIN 1 mg/L each. Stem discs from distal end were less efficient in shoot bud induction. A combination of two cytokinins (BAP and KIN) as a synergistic effect on shoot buds induction from each segment of stem axis. Stem discs in inverted position produced shoot buds from the lower surface, showing strong polarity within the explant. Further, storability and shoot development of sodium alginate encapsulated shoot buds of Curculigo orchioides were tested on half-strength Murashige and Skoog (MS) basal medium fortified with coconut water (10% v/v). The frequency of regeneration from encapsulated shoot buds was affected significantly by concentration of sodium alginate and the duration of exposure to calcium chloride. Shoot buds encapsulated with 2.5% sodium alginate dissolved in MS basal salts solution recorded significantly higher shoot development than other treatments. A relatively short (5 min) incubation with calcium chloride solution provided uniform encapsulation of shoot buds that gave the highest percentage (68%) of shoot development. Encapsulated shoot buds could be stored at 4°C for 50 days without reduction in viability as oppose to non-encapsulated shoot buds, which showed 9.5% viability after 20 days at 4°C. Encapsulated shoot bud developed into normal shoots. Based on the present observations an improved protocol may be developed for the rapid multiplication and conservation of the endangered species—C. orchioides.     

Basipetal Gradient of Axillary Bud Inhibition Along a Rose (Rosa hybridaL.) Stem: Growth Potential of Primary Buds and their Two Most Basal Secondary Buds as Affected by Position and Age

InRosa hybridaL. cv. Ruidriko ‘Vivaldi’®, theeffect of position on growth and development potentials of axillarybuds was investigated by single internode cuttings excised alongthe floral stem and its bearing shoot. The experiments werecarried out in both glasshouses and in a phytotron. The studyfirstly concerned the development of the primary shoot fromthe onset of bud growth until anthesis. The primary shoot wasthen bent horizontally to promote the growth of the two mostproximal secondary buds, the collateral buds, already differentiatedinside the primary bud. They gave rise to basal shoots. In thebasipetal direction, the axillary buds along the floral stemexhibited both an increase in the lag time before bud growthand a decrease in bud growth percentage, demonstrating the existenceof a physiological basipetal gradient of inhibition intrinsicto the buds or due to short range correlations. The same basipetalgradient of inhibition was observed along the floral stem andits bearing shoot, demonstrating that the age of the bud wasnot a major factor in determining the rate of bud growth. Afterbending the primary shoot, the percentage of collateral budgrowth was also affected by the cutting position. The more proximalthe cutting, the lower the sprouting ability of collateral buds.The growth potential of these buds appeared to be already determinedinside the main bud before cutting excision.Copyright 1998 Annalsof Botany Company Axillary bud; basal shoot; cutting; development; endodormancy; growth; paradormancy; position; primary shoot;Rosa hybridaL.; rose; secondary bud; topophysis.     

The Dormancy of Buds of Citrus sinensis (L.) Osbeck Inserted into Rootstock Stems: Factors Intrinsic to the Inserted Bud

Buds of sweet orange, harvested from shoots of different timeof flushing and from different positions along the shoot, wereused to examine whether lack of burst of inserted buds was acharacteristic of the bud. Bursting of inserted buds was significantlyslower in buds taken from (a) older branches (b) shoots producedunder winter conditions, and (c) basal rather than apical budson the same shoot. The slowness to burst when transferred matched a tendency todormancy in buds on shoot segments grown in vitro, suggestingthat the variation in budburst was intrinsic to the bud. Budburstwas correlated with the extent of secondary bud development;the majority of buds from apical regions of the shoot had developeda secondary bud by the time of implantation, but basal budshad not. Adequate vascular connections with the host tissueswere found in both burst and unburst buds. Citrus sinensis (L.) Osbeck, sweet orange, buds, endodormancy, budding     

Histology of the Morphogenic Response in Thin Cell Layer Explants from Vegetative Tobacco Plants

The morphogenic response of thin cell layers (TCLs) from vegetativetobacco (Nicotiana tabacum L.) plants can be directed very preciselyby varying the concentrations of benzyladenine (BA) and -naphthaleneacetic acid (NAA) in the culture medium. Medium containing 1·6µM BA and 0·5 µM NAA was optimal for shootformation, concentrations of 0·5 µM BA and 1·6µM NAA were optimal for the induction of shoots and rootson the same explant, whereas concentrations of NAA higher than16 µM resulted in callus proliferation only. Polarityin the distribution of the shoot buds was observed, i.e. a switchfrom basal to apical shoot formation occurred with increasingNAA concentrations, suggesting basipetal transport of NAA. Histologicalexamination of TCLs on shoot induction medium revealed thatfirst cell divisions occurred within 2 d in cortical cells whichwere directly in contact with the medium along the longitudinalcut surface, and after 2 d in subepidermal cells along the lateraledges of the explants. Individual lateral buds originated fromone subepidermal and one or more epidermal cells, while apicalbuds originated from single subepidermal or cortical cells locateddirectly at the apical end of the explant. After culture ofTCLs for 2-3 d on root/shoot induction medium cells in the regeneration-competentsubepidermis elongated, while on callus induction medium subepidermalcells elongated and dedifferentiated. The regeneration systemas described in this study will be used to identify cells competentfor regeneration as well as for transformation.Copyright 1994,1999 Academic Press Nicotiana tabacum L., tobacco, thin cell layer explants, cell competence, shoot development, polarity     

Development and Growth Potential of Axillary Buds in Roses as Affected by Bud Age

The effect of axillary bud age on the development and potentialfor growth of the bud into a shoot was studied in roses. Ageof the buds occupying a similar position on the plant variedfrom 'subtending leaf just unfolded' up to 1 year later. Withincreasing age of the axillary bud its dry mass, dry-matterpercentage and number of leaves, including leaf primordia, increased.The apical meristem of the axillary bud remained vegetativeas long as subjected to apical dominance, even for 1 year. The potential for growth of buds was studied either by pruningthe parent shoot above the bud, by grafting the bud or by culturingthe bud in vitro. When the correlative inhibition (i.e. dominationof the apical region over the axillary buds) was released, additionalleaves and eventually a flower formed. The number of additionalleaves decreased with increasing bud age and became more orless constant for axillary buds of shoots beyond the harvestablestage, while the total number of leaves preceding the flowerincreased. An increase in bud age was reflected in a greaternumber of scales, including transitional leaves, and in a greaternumber of non-elongated internodes of the subsequent shoot.Time until bud break slightly decreased with increasing budage; it was long, relatively, for 1 year old buds, when theysprouted attached to the parent shoot. Shoot length, mass andleaf area were not clearly affected by the age of the bud thatdeveloped into the shoot. With increasing bud age the numberof pith cells in the subsequent shoot increased, indicatinga greater potential diameter of the shoot. However, final diameterwas dependent on the assimilate supply after bud break. Axillarybuds obviously need a certain developmental stage to be ableto break. When released from correlative inhibition at an earlierstage, increased leaf initiation occurs before bud break.Copyright1994, 1999 Academic Press Age, axillary bud, cell number, cell size, pith, shoot growth, Rosa hybrida, rose     

Micropropagation of Mandevilla moricandiana (A.DC.) Woodson

A protocol was developed for micropropagation of Mandevilla moricandiana (A.DC.) Woodson, a native plant from Brazil. Shoots, obtained from in vitro plantlets were used as source of nodal segments for shoot production from axillary buds. The nodal segments were grown on Murashige and Skoog medium supplemented with different concentrations of 6-benzyladenine and/or indole-3-acetic acid to induce axillary bud elongation. After a 2-mo culture period, the medium supplemented with 1.0 mg?L^?1 6-benzyladenine gave the largest number of nodal segments per explant. The nodal segments obtained from plants developed under these conditions were grown on medium supplemented with different concentrations indole-3-acetic acid, ??-naphthaleneacetic acid, and indole-3-butyric acid. The use of the medium supplemented with indole-3-acetic acid and indole-3-buryric induced shoot elongation and shoot development, formation of basal callus, and/or indirect organogenesis of roots. Following transfer of shoots to soil, the plants with only basal callus showed 10% survival and developed roots from callus, while in vitro-rooted plants had a maximum 40% survival rate ex vitro. Regardless of the auxin added to the rooting medium, the acclimatization period allowed the plants rooted in vitro to develop their shoots fully. The protocol developed here is suitable for the production of shoots and rooted plantlets of M. moricandiana.     

Light cytokinin interactions in shoot formation in epicotyl cuttings of Troyer citrange cultured in vitro

Ilumination did not affect the pathway of shoot regeneration at the cut edges of epicotyl explants of Troyer citrange (Moreira-Dias et al. 2000, 2001), but signigficantly affected the number of developed shoots and the response to exogenous cytokinins. Shoot regeneration at the apical end occurred through a direct organogenic pathway without callus formation. For explants incubated in the light, this regeneration did not require cytokinin addendum, but the number of shoots formed was significantly increased by benzyl adenine, but not by zeatin or kinetin. Incubation in the dark almost suppressed shoot formation at the apical end. The addition of benzyl adenine or kinetin, but not of zeatin, restored shoot formation in the dark to the value obtained in the light. At the basal end of the explants shoot regeneration occurred through an indirect organogenic pathway after the formation of a primary callus. In explants incubated in the light, callus formation and shoot growth was supported by a low (0.5–1 mg l⁻¹) benzyl adenine concentration and by zeatin. Kinetin did not support callus growth. Shoot formation was higher in the presence of benzyl adenine (0.5–1 mg l⁻¹) than of zeatin, but was inhibited by a high (5 mg l⁻¹) benzyl adenine concentration. Incubation in the dark increased callus growth and shoot formation at the basal cut as compared to explants incubated in the light. The three cytokinins tested supported callus growth and shoot formation in the dark, zeatin being the most effective and kinetin the least. In terms of number of shoots developed, the optimum cytokinin addendum depended on the pathway of organogenesis and the conditions of incubation. The maximum number of shoots developed at the apical end was obtained when the incubation was performed in the light in the presence of benzyl adenine. At the basal end, the optimal conditions were incubation in the dark in the presence of zeatin. It was not always possible to define an optimal cytokinin concentration as the curve concentration/response varied from experiment to experiment, which seemed unrelated to the endogenous cytokinin concentration in the explants.     

The apical control of lateral bud development in excised shoot tips of Cuscuta reflexa cultured in vitro

Excised shoot tips of Cuscuta reflexa Roxb. (dodder), a rootless and leafless angiospermic plant parasite, were cultured in vitro for the study of the control of lateral bud development by the apex. In a chemically defined medium lacking hormones, the basal bud alone developed into a shoot. The addition of coconut milk to the growth medium induced the activation of multiple lateral buds, but only a single bud developed further into a shoot. The decapitation of this shoot induced the development of another shoot and the process could be repeated. This showed the controlling effect of the apex in correlative control of bud development. Application of indole-3-acetic acid to the shoot tip explant delayed the development of the lateral bud. Gibberellic acid A₃ induced a marked elongation growth of the explant and reinforced apical dominance. The direct application of cytokinin to an inhibited bud relieved it from apical dominance. A basipetally decreasing concentration gradient of auxin may prevail at the nodes. Bud outgrowth is probably stimulated by cytokinin produced locally in the bud.     

Effects of genotype,explant source and growth regulators on organogenesis in carnation callus

For callus induction, shoot tips and nodal or internodal stem segments of carnation cultivars (Coral, Jaguar, Salome and Sarinah) were grown on MS basal medium with 2,4-dichlorophenoxyacetic acid and kinetin. To achieve organogenesis, calli were transferred onto MS medium without or with growth regulators (indoleacetic acid, naphthaleneacetic acid, indolebutyric acid, kinetin, benzyladenine) in different combinations. Shoot primordia emerged from the subsurface meristemoids of calli, roots developed from the inner callus cells. The effects of genotype, explant source and growth regulators on callus-mediated organogenesis differently manifested themselves in caulogenesis and rhizogenesis, respectively. The number of root-forming calli most of all depended on genotype and least of all on explant source. Unlike rhizogenesis, caulogenesis essentially depended on explant source: internodal calli of all the tested cultivars practically missed the shoot formation ability. The number of caulogenetic calli from apical-nodal segments significantly depended on genotype, but was also affected by growth regulators. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Studies of Growth and Flowering in Picea sitchensis (Bong.) Carr. 2. Initiation and Development of Male, Female and Vegetative Buds

Vegetative shoots from the base of the crown, and from partsof the tree likely to form male or female buds, were collectedfrom 40–years–old trees of Picea sitchensis (Bong.)Carr. throughout the 1973–4 annual growth cycle. The morphologyand growth rates of the terminal buds on these shoots were assessed. Bud scale primordia were formed most quickly in the female position,at an intermediate rate in the male position and most slowlyin the basal vegetative position during April, May and June.In July and early August the apical meristems swelled to formdomes and continued to grow at the same relative rates in themale, female and basal vegetative positions. Reproductive budswere first morphologically distinct in late August and sporangiaappeared in October. Dormancy, defined by the pause in apicalvolume increase, extended from mid-October to mid–March.Young strobili grew much faster than basal vegetative shootsof the same age between mid–March and bud burst in lateApril. Throughout the growth cycle, external changes in budsize reflected changes in size of the apical meristem, youngstrobihis or young vegetative shoot inside the bud. It is proposed that the rate of growth of an apical meristemmay be causally related to the type of bud which subsequentlydevelops from it. Sitka spruce, Picea sitchensis, bud development, morphology, growth of apical dome, flowering     

A rapid in vitro propagation protocol for Piper barberi Gamble, a critically endangered plant

Summary A protocol is described for rapid multiplication of Piper barberi Gamble (Piperaceae) through shoot tip and nodal explant cultures. Nodal explants with a single axillary meristem showed three times better response with respect to shoot proliferation when compared to shoot tip explants. The best shoot proliferation response of nodal explants was observed with a cytokinin combination of N₆-benzyladenine (4.43 μM) and kinetin (2.32 μM), with 88% bud break. The number of shoot initials (2.4) produced per nodal explant was twice the number of shoot initials (1.2) per shoot tip. An average of 6.9±0.58 adventitious shoots were observed from the proximal end of the internodal explants on Mursashige and Skoog (1962) (Ms) basal medium supplemented with N₆-benzyladenine (2.22 μM) and kinetin (0.46 μM). A multiplication rate of 82 shoots per explant could be achieved after 9 wk of subculturing. The in vitro shoots were rooted on one-half and one-quarter MS basal medium. The shoots rooted on one-quarter MS in the dark produced eight roots with an average root length of 3.36 cm and 98% survival. These plants were transferred to the field with a survival rate of 75%.     

Anatomical and ultrastructural analyses of in vitro organogenesis from root explants of commercial passion fruit (Passiflora edulis Sims)

This study aimed to characterize the anatomical events and ultrastructural aspects of direct and indirect in vitro organogenesis in Passiflora edulis. Root explants were cultured on induction medium, supplemented with 4.44 μM 6-benzyladenine. Roots at different stages of development were collected and processed for observation by light microscopy and scanning and transmission electron microscopy. Patterns of direct and indirect regeneration were observed in the explants. During direct organogenesis, the organogenic buds and nodules, formed from meristemoids, originated from the pericycle regions distant from the cut surface. Completely differentiated buds were observed after 20 days of culture. During indirect organogenesis, bud formation occurred via meristemoids at the periphery of the calli, which differentiated from the cortical region of the initial explant. Regardless of the regeneration pattern, the meristemoids had similar ultrastructural characteristics; however, differences were reported in the nuclear shape of the cells of the meristemoids formed directly and indirectly. This study provides important information for enhancing the understanding and characterization of the organogenic process in non-meristematic explants and provides information on the use of roots as explants in genetic transformation protocols for this important tropical species.