Polar transport of 1-naphthaleneacetic Acid determines the distribution of flower buds on explants of tobacco

Upon addition of 1-naphthaleneacetic acid (1-NAA) and benzylaminopurine, flower buds developed on explants from flower stalks of Nicotiana tabacum L. cv Samsun cultured in vitro. At low concentrations of 1-NAA, buds emerged mainly at the basal edge, whereas at high concentrations they developed on the remaining surface. The optimum concentrations for the two groups of buds were 0.45 micromolar and 2.2 micromolar, respectively, and the shapes of the concentration versus response curves were similar. The level of benzylaminopurine in the medium affected neither the shape nor the optimum concentration of these curves. The distribution of the buds over the explants was shown to be caused by polar auxin transport, leading to accumulation at the basal side. First, in the presence of the inhibitors 2,3,5-triiodobenzoic acid and 1-naphthylphthalamic acid, both groups of buds had the same optimum concentration of 1 micromolar 1-NAA. Second, after 6 hours of culture applied 1-NAA had accumulated in the basal part of the explant. In the presence of 1-naphthylphthalamic acid, no transport or accumulation of applied 1-NAA occurred.     

Analysis of Phytohormone Profiles during Male and Female Cone Initiation and Early Differentiation in Long-shoot Buds of Lodgepole Pine

In lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm), cone initiation and gender differentiation are site-specific in long-shoot buds, with female cones in the distal portion and male cones in the proximal portion. By using high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS) in multiple-reaction monitoring (MRM) mode, cytokinins, indole-3-acetic acid (IAA), abscisic acid (ABA), and their selected metabolites were investigated in developing long-shoot buds from multiple genotypes. Spatially, higher concentrations of trans-zeatin riboside (t-ZR) and dihydrozeatin riboside (dhZR) existed in the distal parts of long-shoot buds, whereas concentrations of isopentenyl adenosine (iPA), IAA, ABA glucose ester (ABA-GE), and phaseic acid (PA) were higher in the proximal parts in all investigated genotypes. In long-shoot buds of genotypes with a history of high female cone yield, concentrations of t-ZR and the ratio of zeatin-type to isopentenyl-type cytokinins were higher in the entire buds, whereas dhZR or IAA was higher in either the distal or the proximal part, respectively. In low female cone yielding genotypes, concentrations of c-ZR, iPA, ABA-GE, and PA were higher in both of the parts. Temporally, concentrations of several hormone-related compounds showed obvious changes in late June and late July, prior to male and female cone bud differentiation. This study reveals that the local hormonal status in a long-shoot bud at specific developmental stages may play an important role in gender determination and cone yield.     

Encapsulation of micropropagated buds of six woody species

Regrowth after encapsulation in a sodium alginate matrix of micropropagated buds from six different in vitro proliferated woody species was evaluated. Actinidia deliciosa Liang & Ferguson (kiwifruit), Betula pendula Roth (birch), Crataegus oxyacantha L. (hawthorn), Malus spp. (apple), Rubus spp. (blackberry) and Rubus idaeus L. (raspberry) propagated in vitro were used as bud sources. Encapsulation with sodium alginate and subsequent regrowth on nutrient rich medium was compared to encapsulation with nutrient-enriched alginate capsules followed by regrowth on nutrientless medium. Apical and sub-apical buds of Malus (rootstock M. 27 and cultivar Starkspur Red) were also compared for encapsulation and regrowth ability. All species showed a regrowth after encapsulation, but only if cultured on enriched media. M.27 apical and sub-apical buds showed different regrowth ability after encapsulation with sodium alginate. Applicability of encapsulation of single micropropagated tree buds is discussed.     

Carbon Dioxide and Flowering in Pharbitis nil Choisy

The effects of photoperiod on floral and vegetative development of Pharbitis nil were modified by atmospheric CO₂ concentrations maintained during plant growth. Short day (SD) photoperiods caused rapid flowering in Pharbitis plants growing in 0.03 or 0.1% CO₂, while plants in long day (LD) conditions remained vegetative. At 1 or 5% CO₂, however, flower buds were developed under both the SD and LD photoperiods. Flowering was earliest in the plants exposed to SD at low CO₂ concentrations which formed floral buds at stem node 3 or 4. At high CO₂ concentrations, floral buds did not form until stem node 6 or 7. Both high CO₂ concentrations and LD photoperiods tended to enhance stem elongation and leaf formation.     

Encapsulation of moss buds: an efficient method for the in vitro conservation and regeneration of the endangered moss <Emphasis Type="Italic">Splachnum ampullaceum</Emphasis>

In vitro culture techniques are usually employed for ex situ conservation of endangered plant species. However, encapsulation to preserve threatened bryophytes is scarcely used, and only as a pretreatment prior to cryopreservation. In our study, two different methods of germplasm conservation, involving calcium-alginate encapsulation of moss material, were assessed. The plant material used was gametophyte buds (gametophores) of Splachnum ampullaceum Hedw., a rare species of moss. Moss regeneration was evaluated at different periods of time to examine the efficacy of the technique for moss germplasm conservation. The effects of encapsulation and cold storage on developmental parameters such as protonematal colony diameter, bud length, and number of buds were also studied. The results suggest that moss encapsulation with no prior treatment may be a suitable method for germplasm conservation during long periods of time. With our method survival rates as high as 50% could be reached after 2.5 years of cold storage versus 0% of 24-h cryopreserved beads. This technique together with cold storage, avoiding freezing, may be especially important in desiccation intolerant mosses.     

Cryopreservation of ex-vitro-grown Rosa chinensis ‘Old Blush’ buds using droplet-vitrification and encapsulation-dehydration

Axillary buds from greenhouse-grown plants of Rosa chinensis ‘Old Blush’ were successfully used to establish cryopreservation protocols using both droplet-vitrification and encapsulation-dehydration methods. In droplet vitrification, regrowth occurred after exposure to liquid nitrogen even without pre-culture in the loading solution (LS) before immersion in the plant vitrification solution 2 (PVS2). However, a 20–80 min LS step followed by a short immersion in PVS2 for 3 or 15 min, at 0 °C gave the best regrowth rates (82–86 %). In encapsulation dehydration, the level of dehydration significantly influenced shoot regrowth. The best regrowth rate, 60 %, was obtained at a bead water content of 0.35 g water per g dry weight. These results demonstrate the possibility of using greenhouse plants of rose for cryopreservation by droplet vitrification and encapsulation dehydration.     

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.     

Effect of auxins and cytokinins on plant regeneration from hypocotyls and cotyledons in niger (Guizotia abyssinica Cass.)

Tissue cultures were established from hypocotyl and cotyledonary leaf segments ofGuizotia abyssinica Cass. on MS medium supplemented with various concentrations of auxins (IAA, NAA, IBA or 2,4-D) and cytokinins (KN or BA). Expiants cultured on media with cytokinins or in combination with auxins produced shoot buds. Maximum number of shoot buds (20–25 per culture) were differentiated from cotyledonary leaf segments on medium with 2 mg 1^-1 each of KN and IBA. Rooting of regenerated shoot buds was acheived on medium with NAA. The obtained plantlets were successfully transferred to soil.     

An excellent source of vegetative buds for use in plant hormone studies on apical dominance

When studying the role of plant hormones in the control of growth at apical meristems, it is often difficult to obtain needed amounts of physiologically uniform buds. A source and method are described for obtaining sufficient quantities of large, uniform buds and for the treatment of the buds with indoleacetic acid and kinetin. Buds from the root system of Euphorbia esula L. were grown in Petri plates, with agar suspending the short root sections from which they emanate. Plant hormones are applied by their incorporation in the agar. The effect of various concentrations of indoleacetic acid and kinetin on bud growth was examined.     

In vitro regeneration of adult Pinus sylvestris L. trees

The propagation of adult conifer trees by tissue culture has been studied for the last twenty years, but problems related to the juvenile to adult phase change of trees have limited the practical applications of these tissue culture procedures. This paper describes a micropropagation protocol for the in vitro propagation of mature Scots pine trees. In this study, dormant shoot buds, which had not started to elongate, were collected from twenty-one adult Pinus sylvestris trees (> 15 years old) during the winter. The sampled buds were cut transversely into slices of 0.5 to 1 cm in thickness and were cultured on three types of culture media (DCR, WP and LPm) supplemented with four cytokinins (BA, mT, Tdz and Z), at two different concentrations (25 and 50 µM), except for Tdz, whose concentrations were diluted to 5 µM and 2.5 µM. The evaluated culture media did not show significant differences in the bud organogenesis capacity. In fact, the highest organogenic response was obtained with buds cultured on DCR and WP media and by explants cultured on medium supplemented with 25 µM meta-topolin. This protocol is a successful and efficient biotechnological approach to the micropropagation of adult P. sylvestris trees.     

Encapsulation of internode regenerated adventitious shoot buds of Indian Siris in alginate beads for temporary storage and twofold clonal plant production

This study for the first time demonstrates single bead alginate encapsulation and conversion (multiple shootlets rejuvenation) from adventitious shoot buds (AB) of Albizia lebbeck (L.) Benth. Internodal (IN) segments isolated from field grown 1-year-old plant of A. lebbeck were used for AB induction under in vitro conditions. IN segments incubated on MS medium augmented with 10.0 µM BA exhibited highest adventitious shoot bud induction frequencies (76 %) on all over the surface after 10 weeks of culture. Induced AB were detached from in vitro proliferating cultures and used for encapsulation as an explant to produce large number of synseeds (07–08) from a single IN explant. Four to five AB were encapsulated in a single calcium alginate bead to manage mass propagation, interim storage and germplasm sharing. The finer gel matrix for encapsulation was attained using 3.0 % sodium alginate and 100 mM calcium chloride. Highest percentage of shoot emergence and multiplication (75 %) from synseed was obtained on MS + 10.0 µM BA + 1.0 µM NAA (RM) after 10 weeks of culture. Encapsulated adventitious buds stored at 4 °C for 1–8 weeks (2 months) too showed thriving shoot emergence (68 %) and multiplication in encapsulated AB and development into complete plantlets when returned to RM. Hence, 4–5 encapsulated AB stored at 4 °C, when cultured back to RM also showed shoot induction resulting in up to 10 shoots per synseeds after 10 weeks of culture. Healthy root formation (½ MS + 2.0 µM IBA) and acclimatization were optimized by using previously standardized protocol (Perveen et al. in J For Res 22:47–52, 2011). Genetic stability of synseed-derived plantlets acclimatized under ex vitro was assessed and compared with mother plant using inter-simple sequence repeats (ISSR) analysis. The synthetic seeds have the achievability of being a substitute planting material for the forestry sector in future, especially for the multipurpose plant species.     

Determination of taurine and other acidic amino acids in plants

Micromolar concentrations of taurine (about 10 μmol per kg fresh weight) were obtained in the green tissues of higher plants and in the seeds of Phaseolus vulgaris (about 50 μmol/kg). Fruits in general, and also potatoes and fungal sporophores, contained very little taurine. Phosphoserine and phosphoethanolamine showed much higher concentrations in the seeds of Phaseolus vulgaris and in the winter buds of Pinus sylvestris. Their concentrations changed considerably during the growth of the shoot, while that of taurine was more constant.     

Influence of water deficits on the abscisic Acid and indole-3-acetic Acid contents of cotton flower buds and flowers

A field experiment was conducted during the summer of 1988 to test the hypothesis that water deficit affects the abscisic acid (ABA) and indole acetic acid (IAA) concentrations in cotton (Gossypium hirsutum L.) flower buds in ways that predispose young fruits (bolls) that subsequently develop from them to increased abscission rates. Water deficit had little effect on the ABA content of flower buds but increased the ABA content of flowers as much as 66%. Water deficit decreased the concentrations of free and conjugated IAA in flower buds during the first irrigation cycle but increased them during the second cycle. Flowers contained much less IAA than buds. Water deficit slightly increased the conjugated IAA content of flowers but had no effect on the concentration of free IAA in flowers. Because water deficit slightly increased the ABA content but did not decrease the IAA content of flowers, any carry-over effect of water deficit on young boll shedding might have been caused by changes in ABA but not from changes in IAA.     

Floral determination in axillary buds of Nicotiana silvestris

At anthesis of the terminal flower the developmental fates of axillary buds of the long-day plant Nicotiana silvestris were assessed in situ and in isolation. The in situ developmental fate was assessed by decapitating the plant above the bud in question and letting the bud mature. The developmental fate of isolated buds was assessed by removing the bud from the main axis, rooting it, and letting it mature. The number of nodes below the terminal flower of the mature shoot was indicative of the developmental fate of the bud. Terminal meristems of rooted axillary buds exhibited two patterns of development: (1) Their developmental fate was the same as that of in situ buds at the same node or (2) their developmental fate was the same as that of seed-derived plants. For example, terminal meristems of rooted buds from the fourth node below the inflorescence produced either 15 to 19 nodes or 36 to 40 nodes. In situ fourth buds produced 12 to 14 nodes while seed-derived plants produced 33 to 39 nodes. Terminal meristems of rooted axillary buds that exhibited the same developmental fate as that of in situ buds were determined for floral development. Although determined buds produced a terminal flower, all but one had abnormal inflorescences. That is, in the place of floral branches determined buds produced vegetative branches. Four buds that were not determined for floral development had their shoot tips rooted each time the plant bolted. Only when the plants were allowed to grow without being rerooted did they flower. These results indicate that roots may prevent and/or destabilize floral determination in N. silvestris.     

Shoot Feeding as a Nutrient Acquisition Strategy in Free-Living Psylloids

Shoot feeding by sucking insects is accepted as an adaptation to feeding where plant nutrients are most concentrated and/or of higher quality. Psylloids are an important hemipteran taxon, most of which are free-living and comprise many shoot feeding species, whose nutritional ecology has been largely ignored. I conducted a longitudinal study of Ctenarytaina eucalypti (Maskell) and C. bipartita Burckhardt et al. (Aphalaridae) feeding on eucalypts to document how within-plant (ontogenic) variation in nutritional quality, in particular of free amino acids, determines host suitability and hence the distribution and abundance of nymphs. Nymphs were most abundant within developing apical buds but were not more abundant on branchlets of greater vigour (indicated by rate of extension). Nymphs could be found up to two (C. bipartita) to three (C. eucalypti) alternate leaf pairs distant from apical buds but infrequently and in low numbers; they were never found on older, fully expanded leaves. The position of a leaf on a branchlet (indicative of age) determined its nutritional quality. Younger leaves had higher water contents, lower chlorophyll contents and differed in amino acid (essential and non-essential) composition compared to older leaves. The abundance of C. eucalypti nymphs on expanding leaves and in buds was positively correlated with the concentrations of methionine, valine and threonine in E. globulus leaves at the same or comparable position on a branchlet. The abundance of C. bipartita nymphs was positively correlated with foliar leucine concentrations. Shoot feeding by these two psyllids facilitates access to more concentrated, better quality plant nutrients but may not entirely explain the adaptive significance of their behaviour. The humid microclimate created by the architecture of the hosts’ apical buds protects eggs and nymphs from desiccation and is suggested to have had a significant influence on the evolution of host utilisation strategies of psyllids within this genus.     

Effects of Exogenously Applied Gibberellins and Thidiazuron on Phytohormone Profiles of Long-Shoot Buds and Cone Gender Determination in Lodgepole Pine

In long-shoot buds of lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm), cone-bud initiation and gender differentiation occur in a site-specific manner: female cone buds are normally restricted to the distal portion, whereas male cone buds are located in the proximal portion. Application of a paste containing two plant growth regulators gibberellins A₄ + A₇ (GA_4/7) combined with thidiazuron (TDZ) to long-shoot buds prior to cone-bud gender determination altered endogenous phytohormone profiles and induced female cone-bud formation in the proximal portion of the long-shoot bud, where male cone buds normally occur. Induced cone clusters observed in the following spring were either entirely female or a mixture of both female and male cones. Endogenous phytohormones in the long-shoot bud tissues were quantified by the stable isotope dilution method using high performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple-reaction monitoring mode. Applied GA_4/7 + TDZ led to increased concentrations of endogenous zeatin-type cytokinins, that is, trans-zeatin riboside and dihydrozeatin riboside, whereas concentrations of abscisic acid (ABA) and its catabolite, ABA glucose ester, were decreased, all relative to control, in untreated long-shoot bud tissue. Concentrations of extractable GA₄ and GA₇ declined in long-shoot bud tissues over 4 weeks following treatment with exogenous GA_4/7. This study demonstrates that high levels of endogenous zeatin-type cytokinins, together with reduced levels of ABA, both induced by applied GA_4/7 + TDZ, are positively associated with an increased female cone-bud formation in long-shoot buds.     

Regulation of potato meristem development by jasmonic acid in vitro

The influence of jasmonic acid (JA) on differentiation of meristems of the potato,Solanum tuberosum cv. Vesna, was investigated in vitro. Meristems were grown on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) (10 μM), kinetin (10 μM), gibberellic acid (3 μM), as modified by Bang. Addition of JA in concentrations of 0.5–10 μM increased the number of meristems that developed into buds, particularly in meristems isolated from shoots grown from tubers in the dark. JA had no noticeable effect on meristems from germs grown in light. All added concentrations of JA retarded callus and root formation. The inhibitory effect on rhizogenesis disappeared immediately after transfer of the developed buds to medium without JA.     

Direct somatic embryogenesis and encapsulation of somatic embryos for in vitro conservation of Bacopa monnieri (L.) Wettst

Direct somatic embryogenesis and shoot organogenesis were achieved from leaf explants excised from microshoots of Bacopa monnieri cultured on Murashige and Skoog medium containing N6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of explants differentiated somatic embryos and shoot buds on MS medium supplemented with 12.5 µM BA and 1 µM 2,4-D. The frequency of explants differentiating somatic embryos decreased with increasing concentration of 2,4-D. Light and scanning electron microscopy revealed direct differentiation of somatic embryos and shoot buds from explants, and various developmental stages of the somatic embryos were observed. Somatic embryos and apical shoot tips were encapsulated in sodium alginate gel to produce synthetic seeds. The storage of synthetic seeds produced by encapsulation was studied at 4 and 25?°C (room temperature) for a period of 140 days. Encapsulated somatic embryos were found to retain viability after 140 days of storage at both temperatures, whereas encapsulated apical shoot buds failed to germinate even after 40 days when stored at 4?°C. The viability of synthetic seeds was higher when stored at 25?°C. All amplified markers scored by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) were monomorphic for all the plants produced from synthetic seeds following different periods of storage, thus establishing the clonal fidelity of propagated plantlets.     

Inhibition by Ethylene of Auxin-Promotion of Flower Bud Formation in Tobacco Explants Is Absent in Plants Transformed by Agrobacterium rhizogenes

The in vitro regeneration of flower buds was studied in pedicel explants from tobacco (Nicotiana tabacum L., cv Petit Havana) transformed with Agrobacterium rhizogenes, pRi 1855 (agropine type). At a low concentration (0.1 micromolar) of 1-naphthalene-acetic acid, pedicel strips from phenotypically aberrant plants regenerated two to three times more flower buds than explants from untransformed tobacco. Intermediate bud numbers were observed in transformants with a less extreme phenotype. The results can be explained by an increased sensitivity of the transformed explants to auxin with respect to flower bud regeneration. The effect of transformation on the auxin response is fully accounted for by the absence of a negative interaction of endogenous ethylene with 1-naphthaleneacetic acid, a phenomenon normally encountered in untransformed tissues. Three observations led to this conclusion. Application of 1 micromolar AgNO₃ to untransformed explants increased the number of flower buds to the level observed in transformed tissues but had no effect on transformed pedicel strips; exposure to 10 microliters per liter ethylene strongly reduced the response to auxin at all concentrations in untransformed explants but was almost ineffective in the transformed tissues; and endogenous ethylene synthesis occurred at the same rate in both types of explants.     

Revisiting the fate of buds: size and position drive bud mortality and bursting in two coexisting Mediterranean Quercus species with contrasting leaf habit

Understanding the relationships between bud size and position and bud fate through time is crucial for identifying and subsequently modeling the mechanisms underlying tree architecture. However, there is a lack of information on how bud size drives crown architectural patterns in coexisting tree species. We studied bud demography in two coexisting Mediterranean oak species with contrasting leaf habit (Quercus ilex, evergreen; Q. faginea, deciduous). The main objective was to analyse the effect of bud size on the fate of buds with different positions along the shoot (apical, leaf axillary and scale-cataphyll axillary buds). The number, length and position of all buds and stems were recorded in marked branches during 4 years. Study species presented different strategies in bud production and lifespan. The evergreen species showed greater mortality rate than the deciduous one, which produced larger buds. Bud size and position were highly related since apical buds where longer than axillary ones and bud length declined basipetally along the stem. Apical buds had also higher chances of bursting than axillary ones. Within positions, longer buds presented a higher probability of bursting than shorter ones, although no absolute size threshold was found below which bud bursting was impaired. In Q. ilex, four-year-old buds were still viable and able to burst, whereas in Q. faginea practically all buds burst in their first year or died soon after. Such different bud longevities may indicate contrasting strategies in primary growth between both species. Q. ilex is able to accumulate viable buds for several ages, whereas Q. faginea seems to rely on the production of large current-year buds with high bursting probability under favourable environmental conditions.