Adventitious Bud Development Research Articles

Comprehensive study on in vitro propagation of some imported peach rootstocks: in vitro explant surface sterilization and bud proliferation

The present study was conducted in the Laboratory of Tissue Culture, Horticulture Department, Faculty of Agriculture, Damietta University, Egypt. The objective of this study was to establish a micropropagation protocol suitable for three imported peach rootstocks: Okinawa (P. persica), Nemared (P. persica × P. davidiana) × P. persica), and Garnem (P. dulcis × P. persica) in vitro. The results showed that soaking the explants in sodium hypochlorite (NaOCl) at 20% for 15 min produced the highest responsiveness (82.81%), survival (96.61%), with the lowest mortality (3.14%) and contamination (0.24%). Explants of the Garnem genotype had the best response (89.12%), survival (90.62%), lowest mortality (0.00%), and highest contamination (9.37%) when compared to the other genotypes. In comparison with axillary buds, the shoot tip displayed the highest responsiveness, survival, and death (100, 87.40, and 12.59%, respectively), as well as the least significant contamination (0.00%). Additionally, the percentages of responsive, survived, dead, and contaminated explants at the various collection dates varied significantly. The 6-benzylaminopurine (BAP) concentrations used (3 to 5.0 mg/L) demonstrated similar behavior in terms of in vitro proliferation, with rates of 3.77 to 6.11, 4.33 to 8.88, and 3.33 to 7.44 shoot numbers per explant for the Okinawa, Nemared, and Garnem peach rootstocks, respectively, indicating that the number of shoot proliferations is genotype-dependent. Additionally, using 5.0 mg/L BAP in combination with 0.2 mg/L IBA significantly increased average shoot proliferation (96.29%), number of shoots per explant (7.48), and average leaf number/explant (16.33) compared to the other treatments. Based on these results, adventitious bud development was enhanced during in vitro multiplication of the Okinawa, Nemared, and Garnem peach rootstocks by the synergistic interaction of indole-butyric acid (IBA) and 6-benzylaminopurine (BAP).

Immunolocalization of some arabinogalactan protein epitopes during indirect somatic embryogenesis and shoot organogenesis in leaf culture of centaury (Centaurium erythraea Rafn)

Common centaury (Centaurium erythraea Rafn) is a medicinal plant of great importance for both pharmaceutical and food industries. Distribution of arabinogalactan proteins (AGPs) with specific epitopes recognized by seven monoclonal antibodies (mAbs) was investigated during indirect somatic embryogenesis (ISE) and shoot organogenesis (ISO) in centaury leaf culture. Dynamic changes were observed in localization of JIM4, JIM8, JIM13, JIM15, LM2, LM14, and MAC207 epitopes during somatic embryo (SE) and adventitious bud (AB) development. AGPs responded to all tested mAbs and expressed in numerous meristematic centers that were formed on leaf explants. In globular SEs, the distribution pattern of JIM4, JIM13, JIM15, LM2, and MAC207 epitopes was observed, while during the progression of somatic embryo development, the number of detected AGPs decreased. During formation of ABs, the number of detected AGPs also decreased. In fully formed ABs, only JIM4 and MAC207 were detected. The present study suggests JIM13 antibody as a marker for ISE in centaury leaf culture. These results implicated that AGPs were developmentally regulated during centaury ISE and ISO.

Impact of the Leaf Cut Callus Development Stages of Populus on the Tetraploid Production Rate by Colchicine Treatment

This paper examined the impact of the leaf incision callus developmental status of Populus on the tetraploid production efficiency. Using diploid full-sib progeny [(Populus pseudo-simonii × P. nigra ‘zheyin3#’) × (P. × beijingensis)] as experimental material, leaf explants were gathered from seedlings of five genotypes selected randomly from full-sib hybrid progeny cultured in Murashige and Skoog basal medium with 0.4 mg/L 6-benzyladenine and 0.05 mg/L 1-naphthaleneacetic acid. The morphological and cytological characteristics of the incision callus (from the callus origin and adventitious bud development) were observed and divided into five stages based on the characteristics of the callus. The incision callus from each of the five stages was treated with 30 mg/L colchicine for 3 days. Then, the polyploidy level of the regenerated plants was confirmed by flow cytometry analysis and chromosome number counting. The results indicate that the rate of tetraploid production was significantly correlated with the callus development stage of Populus leaves; the most likely stage for chromosome doubling was Stage II, in which the calli initially formed around the cut end. To validate that callus developmental Stage II was the optimal callus developmental stage for chromosome doubling of diploid full-sib progeny, ten full-sib progeny genotypes were treated with 30 mg/L colchicine for 3 days at Stage II. All ten genotypes of the diploid progeny obtained tetraploid with no mixoploid production; the percentage of tetraploid induction was 7.9–13.2%. There were significant differences in the morphological characteristics of the leaves and roots of diploid and tetraploid plantlets.

Interaction Between Strigolactone and Cytokinin on Axillary and Adventitious Bud Development in Zantedeschia

Interaction Between Strigolactone and Cytokinin on Axillary and Adventitious Bud Development in Zantedeschia Strigolactones are known to be involved in the control of branching, particularly axillary bud outgrowth. However, the effect of strigolactone on adventitious bud development is presently unknown, and the interaction of strigolactone with cytokinin on bud outgrowth has not been fully understood. Using a germination assay, high strigolactone levels were detected in a low-branched cultivar (Best Gold) but was less abundant in a high-branched cultivar (Goldilocks), particularly at an early stage of the growth cycle when branching was not visibly evident. In contrast, the concentration of cytokinins directly correlated with branching, suggesting an antagonistic interaction between strigolactones and cytokinins on branching. Supporting this hypothesis, strigolactone reduced the cytokinin stimulation of both axillary and adventitious shoot number in Zantedeschia grown in vitro. The reduced cytokinin concentration in a phenotypically low branched cultivar may have been associated with the inhibition effect of strigolactones. Hence, the fact that the alteration of branching in Zantedeschia or other horticultural species depends on the hormonal balance between these two hormones is quite likely. The idea may be useful for the generation of plants of desired branching at least within the in vitro system.

In vitro adventitious organogenesis and histological characterization from mature nodal explants of Citrus limon

An efficient and robust system of shoot organogenesis for lemon has been developed as an initial step in the transformation and in vitro mutagenesis protocols. In vitro adventitious organogenesis was studied and histologically characterized using mature nodal explants of two important cultivars of Citrus limon, ‘Verna 51’ and ‘Fino 49’. The effects of several plant growth regulator concentrations, incubation conditions, basal medium, explant type and position along the shoot, and ethylene inhibitors were determined. The presence of 1–3 mg L−1 N 6-benzyladenine, in combination with 1 mg L−1 gibberellic acid, was essential for the development of adventitious buds, with the optimum concentration depending on the cultivar, while organogenesis was lowest when N 6-benzyladenine was used in the medium without gibberellic acid. When the effect of different basal media was studied, the best results for percentage of responsive explants were obtained with Murashige and Skoog medium for both cultivars, while the number of buds per regenerating explant was less than 2 in Woody Plant medium. Attempts were made to identify the type of explant that had the highest regeneration ability using mature shoots of C. limon. When different types of explants (nodal segments with the buds completely removed or internodal segments) were compared, the highest percentage of responsive explants was obtained with nodal segments, while with internodal segments, the regeneration percentage was very poor. Histological analysis showed that nodal segments were more reactive than internodal ones and those structural changes occurred sooner in nodal segments. The existence of a morphogenetic gradient along the shoot was also observed and nodal segments from the apical region exhibited the highest regeneration ability. An initial culture period of 2–4 wk in the dark, depending on the cultivar, clearly improved the regeneration percentage and the number of buds for lemon adult explants. Silver thiosulphate increased the regeneration percentage in adult explants of both cultivars, and the best results were obtained with 20 μM silver thiosulphate.

Histología de la regeneración por organogénesis en Paulownia tomentosa (Paulowniaceae)

Paulownia tomentosa is a fast-growing tree species with a considerable economic potential because of its value for wood as well as its high biomass production, and elevated stress tolerance. The objective of the present study was to evaluate the development of adventitious buds in leaves obtained from four-week-old shoots of P. tomentosa, in order to identify the cells involved in in vitro adventitious bud development. Leaves (proximal halves with the petiole) from the first node were excised from four-week-old micropropagated shoots, and cultured on Murashige and Skoog medium, supplemented with 3% (w/v) sucrose, 0.6% (w/v) Sigma agar, 22.7 microM thidiazuron (TDZ) and 2.9 microM indole-3-acetic acid for two weeks, explants were then transferred to the same medium with 0.44 microM N6-benzyladenine for another four weeks. Five explants were collected daily during the two first weeks in TDZ treatment. A total of 140 samples were processed. Most of the buds developed indirectly from the callus formed in the petiole stub, and they became visible after eight-ten days of culture, although some buds were also observed in the area of the laminar cut at the level of the veins. The first histological changes could be observed after two-three days of culture, with the dedifferentiation of some subepidermal and inner parenchyma cells, which exhibited a large, prominent nucleus, densely-stained cytoplasm and a high nucleus-to-cell area ratio. Proliferation of these cells gives rise to meristemoid formation after seven-ten days of culture. Organized cell division in meristemoids allows the formation of bud primordia that emerged from the explants surface. The progressive structural differentiation of the apical meristem, leaf primordia, and procambium strands, led to formation of complete buds that were observed in the exterior of the explants after 10-15 days of culture. Direct development of buds from cells in the subepidermic and/or epidermic layers were observed on the adaxial surface of the petiole. This protocol may be a useful tool for the application of genetic transformation techniques, as it enables to determine specific regions in the foliar explants where the meristemoids formation will take place, and therefore to determine which cells should be the object of genetic transformation.

In vitro organogenesis from root culture segments of Bixa orellana L. (Bixaceae)

The organogenic potential of root explants derived from cultured seedlings of Bixa orellana L. (annatto) was investigated in response to different incubation conditions and either 4.44 μM 6-benzyladenine, 4.54 μM Thidiazuron, or 4.56 μM Zeatin. Explants cultured in liquid media with agitation generally showed better development of adventitious buds versus explants cultured on semi-solid media. The most adventitious buds developed from explants cultured in liquid media under a 16-h photoperiod. Use of Zeatin and Thidiazuron promoted the development of more adventitious buds than 6-benzyladenine but morphological abnormalities among regenerating shoots and plants were observed. Fewer adventitious buds developed from explants cultured in liquid media supplemented with 6-benzyladenine, but the buds gave rise to the highest percentage of morphologically normal regenerated plants. Histological analyses showed that adventitious buds originated from cell proliferation within the pericycle, opposite the protoxylem poles of the explant. Seedling root tissue is useful for in vitro propagation of B. orellana.

EFFECTS OF PACLOBUTRAZOL AND CHLORMEQUAT ON GROWTH PATTERN AND SHOOT PROLIFERATION OF NORMAL AND VARIANT AECHMEA FACIATA BAKER PLANT REGENERATED IN VITRO

ABSTRACT In vitro propagated Aechmea faciata (clones Hip and Bayes) shoot tips regenerated profusely, but exhibited a high incidence (up to 20%) of variant plants. Some of the variation could be observed in vitro. However, after transplanting, the variant somaclones differed significantly, both morphologically and physiologically, from the parent plants. Aechmea variant somaclones are characterized by the development of several leaves on an elongated stem, assuming a grass-like appearance. The leaves are smaller than those of normal plants, non-waxy, and lack thorns and anthocyanin pigmentation. The growth regulators chlormequat ((2-chloroethyl) trimethyl ammonium chloride) and paclobutrazol (2RS,3RS)-1 -(chlorophenyl)-4, 4-dimethyl-2- (1H-1, 2, 4-triazol-l-yl)pentane-ol) retarded normal and variant plants' growth, and changed their leaf number and size. Paclobutrazol induced waxy leaf formation and promoted a massive development of adventitious buds on Aechmea variant somaclones transplanted to the green...

High efficiency in vitro organogenesis from mature tissue explants of Citrus macrophylla and C. aurantium

A simple and efficient protocol for obtaining organogenesis from mature nodal explants of Citrus macrophylla (alemow) and Citrus aurantium (sour orange) has been developed by optimizing the concentrations of the plant growth regulators, the incubation conditions, the basal medium and by the choice of explant. In order to optimize the plant growth regulator balance, explants were cultured in the regeneration medium supplemented with several N6-benzyladenine (BA) concentrations or with 2 mg l−1 BA in combination with kinetin (KIN) or 1-naphthaleneacetic acid (NAA). The presence of BA was found to be essential for the development of adventitious buds; the best results were obtained using BA at 3 and 2 mg l−1 for alemow and sour orange, respectively. The combination of BA with KIN or NAA in the culture medium decreased the regeneration frequency, with respect to the use of BA alone. The effect of three different basal media was rootstock-dependent. For C. macrophylla the best results were obtained with Woody Plant Medium or Driver and Kuniyuki Walnut Medium (DKW). However, for C. aurantium, although high percentages of regenerating explants were obtained independently of the basal medium used, the highest number of buds per regenerating explants was obtained with DKW medium. Attempts were made to identify the type of explants which had a higher regeneration ability using particular regions along the mature shoots of C. macrophylla. When nodal segments, where the buds were completely removed, and internode segments were compared, the highest percentage of responsive explants was obtained with nodal segments. The existence of a morphogenetic gradient along the shoot was observed and the organogenic efficiency was highest when explants from the apical zone were used. Incubation in darkness for 3 or 4 wk was essential for regeneration process in both rootstocks.

In vitro organogenesis of zucchini squash cv. Caserta

A protocol for the in vitro culture of Cucurbita pepo cv. Caserta was studied, using a cotyledon segment with an attached hypocotyl fragment as an explant. First, to determine the optimal seedling age, explants were collected from 4 to 6-day-old in vitro germinated seedlings and cultured in MS basal medium supplemented with benzylaminopurine (BAP, 4.5 µM), under a 16-h photoperiod at 27ºC. Based on the results obtained, the explants collected from the 4-day-old seedlings were then cultured in MS basal medium supplemented with different concentrations of BAP (0, 1.1, 2.2, 3.3, 4.5, or 5.5 µM) and incubated under a 16-h photoperiod at 27ºC. In vitro organogenesis was most efficient with explants collected from 4-day-old seedlings cultured in medium supplemented with 4.5 µM of BAP. After 4 weeks of incubation the development of adventitious buds at the cotyledon/hypocotyl junction could be observed. These buds were transferred to elongation and rooting medium and the developed plants were acclimatized to greenhouse conditions. The morphogenic process was characterized using light and scanning electron microscopy analyses to confirm the organogenesis. The results showed that this alternate explant is efficient for in vitro culture of zucchini squash cv. Caserta. The protocol will be further examined for future use in genetic transformation experiments in this species.

Direct shoot bud organogenesis and plant regeneration from pre-plasmolysed leaf explants in Catharanthus roseus

A protocol for high-frequency shoot bud regeneration from the leaves of Catharanthus roseus is reported here for the first time. A 60-min pre-plasmolytic treatment of leaf explants in a cell protoplast washing medium containing 13% (w/v) mannitol followed by their plating on a half-strength Murashige and Skoog (MS) medium supplemented with 7.0 mg/l 6-benzyladenine (BA) and 3.0 mg/l α-naphthaleneacetic acid (NAA) resulted in the de novo induction and development of adventitious shoot buds in more than 75% of explants. Histological observations revealed a direct origin of these shoot buds from hypodermal tissue around the mid-rib. The rooting in the regenerated shoots was obtained in the presence of 3.0 mg/l indole-3-butyric acid (IBA) and the rooted plants could be successfully established in soil with a 70% rate of success. The relevance of the developed protocol in terpenoid indole alkaloids pathway engineering at the whole-plant level in C. roseus is discussed.

Potential roles for autophosphorylation, kinase activity, and abundance of a CDK-activating kinase (Ee;CDKF;1) during growth in leafy spurge

Leafy spurge (Euphorbia esula L.) is a deep-rooted perennial weed that propagates both by seeds and underground adventitious buds located on the crown and roots. To enhance our understanding of growth and development during seed germination and vegetative propagation, a leafy spurge gene (Accession No. AF230740) encoding a CDK-activating kinase (Ee;CDKF;1) involved in cell-cycle progression was identified, and its function was confirmed based on its ability to rescue a yeast temperature-sensitive CAK mutant (GF2351) and through in vitro kinase assays. Site-directed mutagenesis of Ee;CDKF;1 indicated that two threonine residues (Thr291 and Thr296) were mutually responsible for intra-molecular autophosphorylation and for phosphorylating its substrate protein, cyclin-dependent kinase (CDK). Polyclonal antibodies generated against the Ee;CDKF;1 protein or against a phosphorylated Ee;CDKF;1 peptide [NERYGSL(pT)SC] were used to examine abundance and phosphorylation of CDKF;1 during seed germination and bud growth. The levels of CDKF;1 were lower in dry or imbibed seeds than in germinating seeds or seedlings. Differences in CDKF;1 were also observed during adventitious bud development; small buds appeared to have greater levels of CDKF;1 than large buds. Similar patterns of CDKF;1 expression were detected with either the polyclonal antibody developed using the CDKF;1 protein or the phosphorylated peptide. These results indicated that Thr291 is constitutively phosphorylated in vivo and associated with Ee;CDKF;1 activity. Our results further suggest that a certain level of CDKF;1 activity is maintained in most tissues and may be an important phenomenon for enzymes that regulate early steps in cell-cycle signaling pathways.

Histological characterization of in vitro adventitious organogenesis in Citrus sinensis

The adventitious bud development was induced in epicotyl segments of Valencia sweet orange (Citrus sinensis L. Osbeck). Seeds were cultured in vitro for three weeks in the dark, followed by one week at a 16-h photoperiod. Epicotyl segments were cultured horizontally for the induction of organogenesis in Murashige and Tucker (1969, MT) culture medium supplemented with 1.0 mg dm-3 benzylaminopurine. Samples were observed by light and scanning electron microscopy from day zero to day 25, when buds were well grown. It was shown that the adventitious buds originated directly from the cambial region on the cut ends of the explants.

Adventitious bud development and regeneration in Tillandsia eizii

Summary The bromeliad Tillandsia eizii is a striking species with large, colorful, and persistent inflorescences that can reach 1m in length. The value of this plant as an ornamental and its importance in cultural and religious activities has led to its overcollection in the wild. Clonal propagation via tissue culture may be a means to repopulate native stands while meeting the demands for this species as an ornamental and ceremonial plant. Adventitious bud proliferation was induced from axenically germinated seedling material. Parameters evaluated were the age of explant material at the time of transfer onto bud-induction medium, the concentration of plant growth regulators, and the period of exposure to induction medium. Light and scanning electron microscopy (SEM) established the origin and development of buds. Twelve-week-old seedling explants rapidly initiated adventitious buds after a 30-d induction period on shoot-initiation medium. Adventitious buds were induced in 40% of the explants placed on media with 2mgl 21 6-benzylaminopurine (BA) (8.88mM) plus 0.1mgl 21 a-naphthaleneacetic acid (NAA) (0.54 mM) with some cultures becoming highly prolific after repeated subculture. Shoots elongated in proliferating cultures, and plants were successfully acclimatized and planted into the greenhouse. The results indicate that tissue culture may be used as a means to propagate this epiphytic bromeliad species, which is being seriously affected by deforestation and habitat destruction. In addition, adventitious bud proliferation can provide a means to propagate superior genotypes.

Morphogenic effect of colchicine inCichorium intybusL. root explants culturedin vitro

A simple and efficient protocol for in vitro shoot regeneration from storage root explants of witloof chicory (Cichorium intybus L.) has been used to study the effect of colchicine at different concentrations, and colchi- cine in combination with kinetin or 2,4-D or with 2,4-D and kinetin, in comparison with kinetin and control (no treatment), upon this type of morphogenic event. The results demonstrated a strong stimulatory effect of colchicine at low concentration on shoot regeneration frequency when compared to the number of shoots formed in the con- trol. This effect was comparable to that of kinetin, a growth regulator that induces shoot differentiation. By contrast, the presence of 2,4-D completely inhibited shoot regeneration. Colchicine at high concentration did not induce shoots, and caused the complete death of the explants. A carefully histological analysis of the explants during the in vitro culture period showed the formation of numerous meristematic zones (precursors of adventitious bud and shoot development) in the parenchyma mass; these meris- tems were present only in the explants subjected to the colchicine and to the other treatments able to stimulate shoot differentiation. Nuclei of different sizes were observed in the explants treated with colchicine at low dose, the largest, probably, polyploids. Polyploid nuclei, in fact, were found in the regenerated plants.

The effect of aminoglycoside antibiotics on the adventitious regeneration from apricot leaves and selection of nptII-transformed leaf tissues

The effect of different concentrations of the aminoglycoside antibiotics, geneticin, paromomycin and streptomycin on adventitious regeneration from leaf explants of apricot was tested to design an alternative procedure for selecting transgenic shoots. Streptomycin and paromomycin reduced shoot regeneration percentage with increasing concentration of antibiotics. Almost a complete inhibition of regeneration was reached when 20μM paromomycin was used, although up to 40 μM streptomycin was necessary to completely inhibit regeneration. Geneticin had a very toxic effect on apricot leaves and regeneration was inhibited at almost all concentrations tested. Addition of kanamycin hastened the development of adventitious buds although silver thiosulfate and not kanamycin was responsible for the observed increase in the consistency of the results from independent experiments. Kanamycin and paromomycin at the concentrations tested improved selection of transformed cells and resulted in a larger number of gfp-expressing regions. Paromomycin at 40 and 25.7 μM kanamycin improved proliferation of transformed tissues as compared with the other antibiotics used and non-selected controls.

Developmental Histology of Organogenic and Embryogenic Tissue in Picea omorika Culture

The histological events during adventitious bud and somatic embryo development in explants from Picea omorika seedlings cultivated in vitro have been examined. Meristematic activity in the superficial layers of cotyledons led to bud primordia formation. Bud primordia had characteristic zonal organization with needle primordia arising on the flanks of the meristem. Somatic embryo initial was formed on the cotyledon surface after an unequal transversal division of a cell. The smaller, apical cell gave rise to apical dome and later on secondary, filamentous suspensor while the larger vacuolated cell presented primary suspensor.

A molecular approach to understanding root bud dormancy in leafy spurge

Leafy spurge is a tenacious perennial weed of the Northern Plains. This plant maintains a perennial growth cycle by controlled production and growth of numerous underground adventitious buds. We are using molecular tools to identify signaling pathways that control underground adventitious bud growth and development in leafy spurge. Toward this end, we have used three techniques to identify genes that are differentially expressed concomitantly with the breaking of quiescence in underground buds of leafy spurge. These techniques include differential display of cDNAs, random cloning and sequencing of genes expressed in growing buds, and microarray technology. To date, we have identified more than 16 genes that are differentially expressed in underground buds of leafy spurge during dormancy break and growth initiation. A detailed expression analysis of these genes will allow them to be grouped by their responses to various signals known to play a role in control of underground bud growth. This information will be used to identify key cis-acting elements involved in the regulation of these genes. How such information on signal transduction processes may be used for developing new weed control strategies by the identification of novel target pathways and development of DNA-based herbicides is presented.

A differentially expressed bZIP gene is associated with adventitious shoot regeneration in leaf cultures of Paulownia kawakamii

Plant regeneration in tissue culture is important for the successful application of transgenic technology in crop improvement. We isolated a bZIP motif containing cDNA (PKSF1) from excised leaf cultures of the fast-growing tree Paulownia kawakamii by a novel subtractive hybridization strategy. PKSF1 codes for a leucine zipper transcription factor and is preferentially expressed during adventitious shoot regeneration. It showed about a sixfold higher level of expression in the shoot-forming tissues compared to the callus-forming cultures of Paulownia. In situ hybridization analysis showed that PKSF1 expression was localized in the developing shoot primordia of the shoot-forming cultures. It was also expressed in the apical meristem and provascular tissues in the vegetative shoot apex. Quantitative polymerase chain reaction results showed that only basal levels of transcripts were present in callus-forming tissues at day 0 and day 10, whereas maximal expression was seen in shoot-forming tissues at day 10, suggesting that PKSF1 is associated with adventitious shoot bud development in Paulownia.

Random sequencing of cDNAs and identification of mRNAs

As a first step toward developing a genomics-based research program to study growth and development of underground adventitious shoot buds of leafy spurge, we initiated a leafy spurge expressed sequence tag (EST) database. From the approximately 2,000 clones randomly isolated from a cDNA library made from a population containing growth-induced underground adventitious shoot buds, we have obtained ESTs for 1,105 cDNAs. Approximately 29% of the leafy spurge EST database consists of expressed genes of unknown identity (hypothetical proteins), and 10% represents ribosomal proteins. The remaining 60% of the database is composed of expressed genes that show BLASTX sequence identity scores of ≥ 80 with known GenBank accessions. Clones showing sequence identity to a Histone H3, a gibberellic acid-responsive gene, Tubulin, and a light-harvesting chlorophyll a/b-binding protein were shown to be differentially expressed in underground adventitious shoot buds of leafy spurge after breaking of dormancy. RNA encoding a putative cyclin-dependent protein kinase (CDK)-activating kinase, a gene associated with cell division, and Scarecrow-like 7, a gene involved in GA signaling, were present at similar levels in dormant and growth-induced underground adventitious shoot buds. These data show how even a small EST database can be used to develop a genomics-based research program that will help us identify genes responsive to or involved in the mechanisms controlling underground adventitious shoot bud growth and development.