Partial shoot reiteration in Wollemia nobilis (Araucariaceae) does not arise from 'axillary meristems'

Background and Aims

Conifers are characterized by the paucity of axillary buds which in dicotyledonous trees usually occur at every node. To compensate, conifers also produce ‘axillary meristems’, which may be stimulated to late development. In juvenile material of Wollemia nobilis (Araucariaceae: Massart''s model) first-order (plagiotropic) branches lack both axillary buds and, seemingly, axillary meristems. This contrasts with orthotropic (trunk) axes, which produce branches, either within the terminal bud or as reiterated orthotropic axes originating from axillary meristems. However, plagiotropic axes do produce branches if they are decapitated. This study investigated how this can occur if axillary meristems are not the source.

Methods

The terminal buds of a series of plagiotropic branches on juvenile trees were decapitated in order to generate axillary shoots. Shoots were culled at about weekly intervals to obtain stages in lateral shoot development. Serial sections were cut with a sliding microtome from the distal end of each sample and scanned sequentially for evidence of axillary meristems and early bud development.

Key Results

Anatomical search produced no clear evidence of pre-existing axillary meristems but did reveal stages of bud initiation. Buds were initiated in a group of small starch-rich cortical cells. Further development involved de-differentiation of these small cells and the development of contrasting outer and inner regions. The outer part becomes meristematic and organizes the apex of the new branch. The inner part develops a callus-like tissue of vacuolated cells within which vascular cambia are developed. This kind of insertion of a branch on the parent axis seems not to have been described before.

Conclusions

Axillary meristems in Wollemia characterize the leaf axils of trunk axes so that the origin of reiterated shoots is clear. Plagiotropic axes seemingly lack axillary meristems but still produce axillary branches by distinctive developmental processes. These observations demonstrate limited understanding of branch initiation in trees generally.     

DIFFERENTIATION OF LATERAL SHOOTS AS THORNS IN ULEX EUROPAEUS

Ulex europaeus is a much-branched shrub with small, narrow, spine-tipped leaves and axillary thorn shoots. The origin and development of axillary shoots was studied as a basis for understanding the changes that occur in the axillary shoot apex as it differentiates into a thorn. Axillary bud primordia are derived from detached portions of the apical meristem of the primary shoot. Bud primordia in the axils of juvenile leaves on seedlings develop as leafy shoots while those in the axils of adult leaves become thorns. A variable degree of vegetative development prior to thorn differentiation is exhibited among these secondary thorn shoots even on the same axis. Commonly the meristems of secondary axillary shoots initiate 3–9 bracteal leaves with tertiary axillary buds before differentiating as thorns. In other cases the meristems develop a greater number of leaves and tertiary buds as thorn differentiation is delayed. The initial stages in the differentiation of secondary shoot meristems as thorns are detected between plastochrons 10–20, depending on vigor of the parent shoot. A study of successive lateral buds on a shoot shows an abrupt conversion from vegetative development to thorn differentiation. The conversion involves the termination of meristematic activity of the apex and cessation of leaf initiation. Within the apex a vertical elongation of cells of the rib meristem initials and their immediate derivatives commences the attenuation of the apex which results in the pointed thorn. All cells of the apex elongate parallel to the axis and proceed to sclerify basipetally. Back of the apex some cortical cells in which cell division has persisted longer differentiate as chlorenchyma. Although no new leaves are initiated during the extension of the apex, provascular strands are present in the thorn tip. Fibrovascular bundles and bundles of cortical fibers not associated with vascular tissue differentiate in the thorn tip and are correlated in position with successive incipient leaves in the expected phyllotactic sequence, the more developed bundles being related to the first incipient leaves. Some secondary shoots displayed variable atypical patterns of meristem differentiation such as abrupt conversion of the apex resulting in sclerification with limited cell elongation and small, inhibited leaves. These observations raise questions concerning the nature of thorn induction and the commitment of meristems to thorns.     

Determination for inflorescence development is a stable state,separable from determination for flower development in Pisum sativum L. buds

Terminal meristems of Pisum sativum (garden pea) transit from vegetative to inflorescence development, and begin producing floral axillary meristems. Determination for inflorescence development was assessed by culturing excised buds and meristems. The first node of floral initiation (NFI) for bud expiants developing in culture and for adventitious shoots forming on cultured meristems was compared with the NFI of intact control buds. When terminal buds having eight leaf primordia were excised from plants of different ages (i.e., number of unfolded leaves) and cultured on 6-benzylaminopurine and kinetin-supplemented medium, the NFI was a function of the age of the source plant. By age 3, all terminal buds were determined for inflorescence development. Determination occurred at least eight nodes before the first axillary flower was initiated. Thus, the axillary meristems contributing to the inflorescence had not formed at the time the bud was explanted. Similar results were obtained for cultured axillary buds. In addition, meristems excised without leaf primordia from axillary buds three nodes above the cotyledons of age-3 plants gave rise to adventitious buds with an NFI of 8.3 ±0.3 nodes. In contrast seed-derived plants had an NFI of 16.5 ±0.2. Thus cells within the meristem were determined for inflorescence development. These findings indicate that determination for inflorescence development in P. sativum is a stable developmental state, separable from determination for flower development, and occurring prior to initiation of the inflorescence at the level of meristems.     

Developmental shoot morphology in Phyllocladus (Podocarpaceae)

TOMLINSON, P. B., TAKASO, T. & RATTENBURY, J. A., 1989. Developmental shoot morphology in Phyllocladus (Podocarpaceae). Shoot architecture in the adult phase of Phyllocladus is established by a succession of units of extension that develop a system of permanent axes supporting photosynthetic units (phylloclades) each of which represents a branch complex with three branch orders. Seedlings have needle foliage leaves comparable to those of other conifers, but in adult plants all leaves are ephemeral, non-photosynthetic scales that for the most part subtend no axillary buds. Once rhythmic growth (usually seasonal) is established in the adult phase, each increment produces a whorl of phylloclades so that a regular tiered arrangement develops, with the tiers progressively reduced on outer units. In the resting terminal bud of permanent axes only scale primordia are present; with bud burst and beginning of extension of the unit the phylloclades are produced by syllepsis and complete the initiation and expansion of all axis orders in the short flushing cycle. Segments retain strict distichy throughout, but in a dorsiventral and not a lateral plane. Phylloclades may be either determinate, when the apex of the first-order axis develops as a terminal flattened segment, or indeterminate, when the apex retains radial symmetry and forms a resting bud that can continue axis extension as a permanent shoot in subsequent years. A phylloclade consequently only produces flattened lateral segments in its season of initiation. Reiteration from reserve buds is not possible, because none are produced in the adult phase, but is possible from detached meristems formed in the axils of needle leaves on juvenile shoots. Reiteration in the adult phase is thus possible only by axis dedifferentiation, that is, change from plagiotropy to orthoptropy. The distinctive massive vascular connection of the phylloclade is made possible by syllepsis. In this way the normal structural constraints of elaborate appendage development in conifers is fully overcome.     

Elimination of five viruses from sugarcane using in vitro culture of axillary buds and apical meristems

Procedures were developed for the in vitro elimination of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Sugarcane streak mosaic virus (SCSMV), Sugarcane yellow leaf virus (SCYLV) and Fiji disease virus (FDV) from infected sugarcane. In vitro shoot regeneration, elongation and virus elimination through meristem tissue culture originating from both apical and axillary shoots were compared. The average rates of regeneration and elongation from apical meristem tissues were 91 and 66%, respectively, with the virus-free rate among elongated shoots ranging from 61–92%. Mature axillary buds were cultivated in vitro to produce axillary shoots, from which meristem tissues were excised and cultured. These meristem tissues regenerated (77–100%) and elongated (55–88%) in culture medium at approximately the same rate as the apical meristems. The average virus elimination rate was 90% among elongated shoots derived from mature axillary buds. All five viruses can be eliminated by meristem tissue culture from both apical and axillary shoots using a standardized procedure. The overall average efficiency of virus-free plant production was 45 and 58% from apical and axillary shoots, respectively. There were no significant differences for shoot induction or virus elimination when the meristems were harvested from either the apical or the axillary shoots. This is the first report of SrMV or SCSMV elimination from sugarcane, as well as elimination of any mixed virus infections. This new method of harvesting meristems from axillary buds greatly expands the amount of material available for therapeutic treatments and thereby increases the probability of eliminating viruses from infected sugarcane.     

Influence des racines sur le développement végétatif ou floral des bourgeons cotylédonaires chez le Scrofularia arguta: role possible des cytokinines

Influence of roots on the vegetative or floral development of cotyledonary buds of Scrofularia arguta Sol.: A possible cytokinin role. This study shows that the presence of “nonabsorbing roots” insures a vegetative development of cotyledonary buds cultured in vitro whereas buds growing without roots produce flowers early. In the same way, roots suppress floral expression of axillary meristems of the same cotyledonary buds and induce these buds to vegetative functioning. Various trophic modifications in the culture medium are ineffective on non-rooted buds as also are gibberellin As and adenine. On the contrary, several cytokinins (kinetin, benzyladenine and zeatin) exert the same influence as roots. These results suggest that roots regulate meristematic functioning through cytokinins.     

Localization of axillary meristems of short day plantChenopodium rubrum L.

Axillary meristems of short day plantChenopodium rubrum are localized as caulinar, foliar or axillar. The localization of axillary meristems and axillary buds of 14 day old plants varied in similar pattern as in other plant species so far investigated: after several nodes with foliar axillary meristems the caulinar ones were produced. However, unlike in other species, in C.rubrum a very high percentage of caulinar meristem is produced also on the first node. In this case, like in the case of its later differentiation at higher nodes, the formation of caulinar meristem is confined also to the vegetative state. It was found that the caulinary position coincides with higher responsiveness to photoperiodic induction. The developmental significance of such behaviour is discussed.     

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.     

Florigen is involved in axillary bud development at multiple stages in Arabidopsis

The wide variety of plant architectures is largely based on diverse and flexible modes of axillary shoot development. In Arabidopsis, floral transition (flowering) stimulates axillary bud development. The mechanism that links flowering and axillary bud development is, however, largely unknown. We recently showed that FLOWERING LOCUS T (FT) protein, which acts as florigen, promotes the phase transition of axillary meristems, whereas BRANCHED1 (BRC1) antagonizes the florigen action in axillary buds. Here, we present evidences for another possible role of florigen in axillary bud development. Ectopic overexpression of FT or another florigen gene TWIN SISTER OF FT (TSF) with LEAFY (LFY) induces ectopic buds at cotyledonary axils, confirming the previous proposal that these genes are involved in formation of axillary buds. Taken together with our previous report that florigen promotes axillary shoot elongation, we propose that florigen regulates axillary bud development at multiple stages to coordinate it with flowering in Arabidopsis.     

Developmental events in differentiating floral buds of four olive (Olea europaea L.) cultivars during late winter to early spring

The olive tree (Olea europaea L.) is a very important evergreen fruit tree because of the high interest of olive oil and table olives in the human diet. Differentiation of olive floral buds during winter is strictly related to flowering during spring and finally to fruit production during autumn–winter. In order to determine which are the developmental events in differentiating olive floral buds we studied in four olive cultivars “Amfissis”, “Kalamon”, “Manzanillo” and “Chalkidikis” the anatomical progress of the ontogeny using median longitudinal sections of floral buds (cutting odd nodes), in weekly intervals from early February (initiation of differentiation) to mid-April (one week after floral bud burst) keeping the cultivars under the same environmental conditions. At the same time, we determined the changes in their water extractable proteins, as an index of the meristematic activity. At the beginning of differentiation, floral buds consisted of the apical and one pair of developed (odd) axillary meristems which were covered with the corresponding bracts. Floral buds showed a slow gradual enlargement in all cultivars. The sequence of the developmental events (initiation of intense mitotic activity in the apical and the first axillary meristems, initiation of mitotic activity in the second axillary meristem, development of the third pair of bracts (except from cv. Chalkidikis), appearance of mitotic activity in the third axillary meristem, swelling of apical and axillary meristems and appearance of primordial whorls) are presented comparatively. All cultivars showed similar ontogenetic trends but there were temporal differences in the initiation, the progress and the completion of the process. Cv. Amfissis initiated differentiation one week later compared to the other cultivars, also delaying the development of the second and the third axillary meristems. On the other hand, cvs. Kalamon and Chalkidikis delayed one week to complete the process when compared to cvs. Amfissis and Manzanillo.     

An efficient mass propagation system for cassava (Manihot esculenta Crantz) based on nodal explants and axillary bud-derived meristems

Nodes from 3- to 5-week-old in vitro plants of different cassava cultivars were cultured for 2–3 days on solid Murashige and Skoog basal medium supplemented with cytokinin to induce the enlargement of axillary buds. Subculture of these buds on the same medium resulted in multiple shoot formation within 4–6 weeks. Of the four cytokinins tested (6-benzylaminopurine (BAP), thidiazuron (TDZ), zeatin, and kinetin), BAP induced shoot development most efficiently. The best results were obtained with cultivar TMS 30555, in which 63% of the explants each produced at least 25 shoots on medium with 10 mg/l BAP. In cultivars that did not produce shoots, the addition of the surfactant Pluronic F-68 (2% wt/vol) raised the percentage of explants forming at least 5 shoots from 0 to 20–60%. Axillary buds were also used to dissect meristems and test their ability to regenerate into shoots. Shoot formation from meristems of six different cultivars was observed after preculture on medium with 5 mg/l BAP followed by transfer to 10 mg/l BAP.Abbreviations MS Murashige and Skoog - BAP 6-Benzylaminopurine - TDZ Thidiazuron     

CYTOKININ- AND GIBBERELLIC ACID-INDUCED EFFECTS ON THE STRUCTURE AND METABOLISM OF SHOOT APICAL MERISTEMS IN OPUNTIA POLYACANTHA (CACTACEAE)

The dormant axillary buds of Opuntia polyacantha can be activated by either cytokinins or gibberellic acid. Under the influence of benzylaminopurine (BAP), the axillary bud meristem increases greatly in size and becomes mitotically active. The primordia produced by the meristem develop as normal photosynthetic leaves. Gibberellic acid (GA) also causes the meristem to become mitotically active, but the meristem does not increase in size. The primordia produced under the influence of GA develop as normal cactus spines. Leaf-producing meristems and spine-producing meristems have the same zonation, despite the differences in size. The meristems are composed of a uniseriate tunica, a central mother cell zone, peripheral zone, and a pith rib meristem. The mitotic activity of each of the zones in the leaf-producing meristem differs significantly from the mitotic activity of the corresponding zones in the spine-producing meristem.     

Axillary meristems and the development of epicormic buds in wollemi pine (Wollemia nobilis)

Intact trees of Wollemia nobilis Jones, Hill and Allen (Araucariaceae) routinely develop multiple coppice shoots as well as orthotropic epicormic shoots that become replacement or additional leaders. As these are unusual architectural features for the Araucariaceae, an investigation was made of the axillary meristems of the main stem and their role in the production of epicormic and possibly coppice shoots. Leaf axils, excised from the apex to the base of 2-m-high W. nobilis plants (seedling origin, ex situ grown), were examined anatomically. Small, endogenous, undifferentiated (no leaf primordia, no vascular or provascular connections) meristems were found in the axils from near the shoot apex. In the more proximal positions about half the meristems sampled did not differentiate further, but became tangentially elongated to compensate for increases in stem diameter. In the remaining axils the meristems slowly developed into bud primordia, although these buds usually developed few leaf primordia and their apical 'domes' were wide and flat. Associated vascular development was generally restricted to provascular dedifferentiation of the cortical parenchyma, with the procambium usually forming a 'closed loop' that did not extend back to the secondary vascular tissues. Development of the meristems was very uneven with adjacent axils often at widely differing stages of development into buds. The study shows that, unlike most conifers, W. nobilis possesses long-lived meristematic potential in most, if not all, leaf axils. Unlike other araucarias that have been investigated, many of the meristems in the orthotropic main stem will slowly develop into bud primordia beneath the bark in intact plants. It appears likely that this slow but continued development provides a ready source of additional or replacement leaders and thus new branches and leaves.     

Gene expression polymorphisms and ESTs associated with gravitropic response of subterranean branch meristems and growth habit in Leymus wildryes

Negatively orthogeotropic (NOGT) tiller and diageotropic (DGT) rhizome meristems develop from the same type of lateral axillary meristems and phytomer structure. Although subterranean NOGT and DGT buds appear similar, they display different responses to gravity and perhaps other cues governing branch angle and overall growth habit (GH). Leymus wildryes show remarkable variation in GH and include some of the largest native grasses in western North America. Previous studies detected GH QTLs on homoeologous regions of LG3a and LG3b controlling differences between caespitose Leymus cinereus and rhizomatous Leymus triticoides allotetraploids. Heterologous barley and wheat microarrays in conjunction with bulk segregate analysis were used to find gene expression polymorphisms associated with GH QTLs. Approximately 34% and 25% of the probe sets showed detectable signals on the barley and wheat arrays, respectively. Overall gene expression patterns of NOGT and DGT meristems were remarkably similar, consistent with the assertion that Leymus NOGT and DGT buds develop from homologous meristems. Only 28 and 27 genes on barley and wheat gene chips, respectively, showed more than twofold differential expressions between NOGT and DGT tissues. One expression polymorphism genetically mapped in the Leymus LG3 rhizome QTL region.     

Cryopreservation of in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation vitrification

 Alginate-coated meristems from in vitro-grown axillary buds of mint (Mentha spicata L.) were successfully cryopreserved by vitrification. Excised meristems from nodal segments cold hardened at 4  °C for 3 weeks were encapsulated and osmoprotected by a mixture of 2 M glycerol plus 0.4 M sucrose. These meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 h at 0  °C prior to a plunge into liquid nitrogen. Successfully encapsulated vitrified meristems developed shoots within a week after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 90%. This procedure was successfully applied to other Mentha species. It was also confirmed that encapsulated vitrified meristems produced a much higher rate of shoot formation than the encapsulated dried meristems. Thus, this revised encapsulation vitrification method appears promising for the cryopreservation of mint and other germplasm. Received: 24 November 1998 / Revision received: 8 February 1999 / Accepted: 26 February 1999     

Somatic embryogenesis from the axillary meristems of peanut (Arachis hypogaea L.)

Developmental anomalies in the plumule meristem of peanut (Arachis hypogaea L.) somatic embryos resulted in poor shoot differentiation and reduced plant recovery. Existing meristems with caulogenic potential have never been tested for embryogenesis in peanut. The present experiment was designed to test the mature zygotic embryo axis derived plumule with three meristems for somatic embryogenesis. Embryogenic masses and embryos developed from the caulogenic meristems in the axils. Exposure of 2 weeks in primary medium with 90.5 μM 2,4-D suppressed the shoot tip differentiation temporarily which then regained the ability to form the shoot on withdrawal of 2,4-D. Exposure of 4 weeks in primary medium with 90.5 μM 2,4-D suppressed the shoot tip differentiation irreversibly. No shoot formation was noted from the tips in any of the cultures which were in secondary medium with 13.6 μM 2,4-D. Development of somatic embryos directly from axillary meristems was confirmed histologically. Conversion frequency of these embryos was 11%. Thus, in this report, we describe a method to obtain somatic embryos from the determined organogenic buds of the axillary meristem, by culturing the nodal explant vertically on embryo induction medium. It also displays the possibility of obtaining both embryogenic and organogenic potential in two parts of the same explant simultaneously. The possibility of extending this approach for genetic transformation in in vivo system through direct DNA delivery or Agrobacterium injection in meristems can also be explored. Using Agrobacterium rhizogenes, we have demonstrated the possibility of gene transfer in the axillary meristems of seed-derived plumule explant.     

THE ORIGIN AND DEVELOPMENT OF INDUCED BRANCHES IN HELIANTHUS

The development of buds and their vascular connections are described for Helianthus annuus and H. bolanderi. Bud meristems of H. annuus usually become isolated by parenchymatization of the bud traces in the cortical zone. If the buds are induced to grow as a result of decapitation of the terminal meristem, a continuous range between typical primary connections and pseudo-adventitious connections are made between the main axis and the lateral buds. Considerable growth of the branches occurs within 48 hours after decapitation. Axillary buds of H. bolanderi grow continuously, and both the meristem and vascular system of the buds are derived directly from the apical meristem of the shoots.     

EXPERIMENTAL STUDIES OF THE SHOOT APICAL MERISTEM OF SEED PLANTS. I. MORPHOLOGICAL AND CYTOCHEMICAL EFFECTS OF IAA APPLIED TO THE EXPOSED MERISTEM OF LUPINUS ALBUS

Application of 1.5 μg indoleacetic acid (IAA) in a lanolin droplet to the exposed apical meristem of Lupinus albus seedlings caused: (1) axillary buds to form closer to the apex than normal, (2) displacement of primordia formed during the first two plastochrons following treatment, and (3) significant increases in concentration of RNA, protein, and unsaturated lipids in the meristems. Primordial displacement tended to be random relative to the site of the treatment, which may be a feature common to dicotyledonous plants exhibiting spiral phyllotaxis. That IAA conferred initiation site capabilities to all of the peripheral zone for a short time was indicated by (2) and (3) above, and by decreases in concentrations of the observed compounds toward control levels after the second plastochron following treatment. Effects of IAA on RNA suggest that nucleic acid metabolism, and possibly gene action, was involved in the response. Kinetin or gibberellic acid had no apparent morphogenetic effect on Lupinus meristems.     

NATURAL REGENERATION OF GINKGO BILOBA FROM DOWNWARD GROWING COTYLEDONARY BUDS (BASAL CHICHI)

This study describes the origin and early development of a distinct organ of clonal regeneration in Ginkgo biloba, the basal chichi. These aggregates of suppressed shoot buds originate from superficial meristems located in the cotyledonary axils of all Ginkgo seedlings as part of their normal ontogeny. Within 6 wk of germination these buds become embedded in the cortex of the stem, and their subsequent growth and development occurs below the surface of the bark. When stimulated by some traumatic event that damages the seedling axis, one of these embedded cotyledonary buds usually grows down from the trunk to form a woody, rhizomelike basal chichi which, under appropriate conditions, is capable of generating both aerial shoots and adventitious roots. Vegetative regeneration by means of basal chichi has not only contributed to the long-term persistence of G. biloba in the forests of China, but may also have played a role in the remarkable survival of the genus since the Cretaceous.