Bud Regeneration Research Articles

Study of senescence in old cultures of the Bactris gasipaes Kunth in vitro

The in vitro propagation is a technique that is used for the large-scale generation of stable genotypes that are free of disease. Satisfactory propagation has been obtained using Bactris gasipaes Kunth through the direct in vitro regeneration of adventitious buds and somatic embryos, which is likely because of the pluri—and totipotency of the preprocambial cells, which reduces the probability of genetic instability. The consequences of the prolonged period of in vitro cultivation of perennial species such as the peach palm are not clear, as well as the senescence process has only been investigated in species originating from callus structures. The senescence of peach palm clones established and cultivated in vitro for 8 years through direct morphogenic methods (in vitro regeneration of adventitious buds without callus formation) was investigated in the leaves, roots and stem bases using histological, histochemical and ultrastructural analyses, in addition to the TUNEL reaction, to detect programmed cell death. These data from old cultures (8 years cultured) were compared to similar data from plants of the same species that had been established and cultivated in vitro for 1 year (young cultures). These analyses demonstrated the existence of intensive programmed cell death in the various tissues of the old cultures. The in vitro cultivation of peach palm for long periods promoted generalized senescence and probable aging of clones.

Transcriptional regulators in the Hippo signaling pathway control organ growth in Xenopus tadpole tail regeneration

The size and shape of tissues are tightly controlled by synchronized processes among cells and tissues to produce an integrated organ. The Hippo signaling pathway controls both cell proliferation and apoptosis by dual signal-transduction states regulated through a repressive kinase cascade. Yap1 and Tead, transcriptional regulators that act downstream of the Hippo signaling kinase cascade, have essential roles in regulating cell proliferation. In amphibian limb or tail regeneration, the local tissue outgrowth terminates when the correct size is reached, suggesting that organ size is strictly controlled during epimorphic organ-level regeneration. We recently demonstrated that Yap1 is required for the regeneration of Xenopus tadpole limb buds (Hayashi et al., 2014, Dev. Biol. 388, 57–67), but the molecular link between the Hippo pathway and organ size control in vertebrate epimorphic regeneration is not fully understood. To examine the requirement of Hippo pathway transcriptional regulators in epimorphic regeneration, including organ size control, we inhibited these regulators during Xenopus tadpole tail regeneration by overexpressing a dominant-negative form of Yap (dnYap) or Tead4 (dnTead4) under a heat-shock promoter in transgenic animal lines. Each inhibition resulted in regeneration defects accompanied by reduced cell mitosis and increased apoptosis. Single-cell gene manipulation experiments indicated that Tead4 cell-autonomously regulates the survival of neural progenitor cells in the regenerating tail. In amphibians, amputation at the proximal level of the tail (deep amputation) results in faster regeneration than that at the distal level (shallow amputation), to restore the original-sized tail with similar timing. However, dnTead4 overexpression abolished the position-dependent differential growth rate of tail regeneration. These results suggest that the transcriptional regulators in the Hippo pathway, Tead4 and Yap1, are required for general vertebrate epimorphic regeneration as well as for organ size control in appendage regeneration. In regenerative medicine, these findings should contribute to the development of three-dimensional organs with the correct size for a patient’s body.

Regeneration of Carthamus tinctorius from Jimsar

ObjectiveTo investigate the factors significantly affecting the shoot buds regeneration of Carthamus tinctorius from Jimsar (CTJ). MethodsThrough tissue culture experiments, the influences of culture temperature, illuminance, relative humidity, explants type, seedlings age, and plant growth regulators added in culture media on in vitro regeneration of CTJ were investigated. ResultsThe culture temperature was set at 24 °C in daytime and 16 °C at night, illuminance at 9000 lx, and 60% relative humidity were suitable for CTJ regeneration. Cotyledons excised from 6–8 d old seedlings were more responsive than even-aged euphyll, hypocotyl, and root explants because of inducing adventitious buds. The highest percentage of regenerated shoots (79.1%) with about five adventitious buds per responding explant was obtained from MS basal medium containing 12.0 mg/L TDZ, 2.5 mg/L IBA, and 1.5 mg/L 2-ip (No. 14 medium). Regenerated shoot buds (80%) could elongate successfully after 1–2 weeks transferred to shoot elongation medium. ConclusionSuitable factors of shoot buds regeneration for CTJ were determined. It may also make a useful reference for regeneration researches of other C. tinctorius varieties in China.

Effect of Adenine Sulphate Interaction on Growth and Development of Shoot Regeneration and Inhibition of Shoot Tip Necrosis Under In Vitro Condition in Adult Syzygium cumini L.—a Multipurpose Tree

An efficient method for cloning Syzygium cumini (above 40years old) through mature nodal segments has been successfully developed and that could be exploited for large-scale production of this valuable multipurpose tree. Nodal segments from mature tree were taken as explants and cultured on MS basal medium with different cytokinins (BA, Kin, AdS). The application of BA proved to be the best responsive cytokinin for the induction of shoot buds and shoots, but the proliferated shoots exhibited slower and stunted growth accompanied with abscission of leaves and shoot tip necrosis (STN). The problem of leaf abscission and STN was considerably reduced by the application of an adjuvant, adenine sulphate (AdS) in the optimal medium which led to the production of a maximum of 14 shoots. Further improvement in shoot bud regeneration and improved growth pattern of the regenerating tissue was obtained on the media comprised of MS + BA (10μM) + GA3 (2.5μM). A total number of 15 shoots with mean shoot length of 5.9cm was obtained. The healthy elongated shoots were then rooted on MS basal augmented with NAA (5μM). The plantlets obtained were healthy and were successfully acclimatized and transferred under field condition with 70% survival rate.

Differential Perception of Caffeine Bitter Taste Depending on Smoking Status

Taste impairment may be associated with tobacco smoking. We assessed taste recognition and intensity among healthy middle-aged current smokers (N = 94, 21 %), former smokers (N = 48, 11 %) and non-smokers (N = 309, 69 %) recruited on a voluntary basis among hospital staff. By means of a whole-mouth gustatory test, participants tasted one concentration of the four basic tastes (NaCl 34 mM, sucrose 58 mM, acetic acid 60 mM, caffeine 1.5 mM for salty, sweet, sour and bitter tastes, respectively) and completed a questionnaire for taste recognition and intensity (ranging from 0 to 10). The recognition of salty, sweet and sour tastes was not influenced by smoking status. Bitter taste recognition was wrong among 13.4, 19.8 and 26.5 % of non-smokers, current smokers and former smokers, respectively (p = 0.043). The adjusted odds ratio (95 % confidence interval) of correct bitter taste recognition was 0.31 (0.14–0.69) among former and 0.74 (0.35–1.55) among current smokers (p = 0.016), compared to non-smokers while adjusting for gender, age, year of assessment and bitter taste intensity. The distribution of caffeine’s bitter taste intensity was bimodal regardless of the smoking status. The differential perception of caffeine’s bitter taste by current and former smokers is likely to be caused by a toxic process. As taste impairment persists in former smokers, the bioaccumulation of some tobacco/combustion products might be responsible for the disequilibrium in taste buds regeneration.

Yap1, transcription regulator in the Hippo signaling pathway, is required for Xenopus limb bud regeneration

The Hippo signaling pathway is conserved from insects to mammals and is important for multiple processes, including cell proliferation, apoptosis and tissue homeostasis. Hippo signaling is also crucial for regeneration, including intercalary regeneration, of the whole body in the flatworm and of the leg in the cricket. However, its role in vertebrate epimorphic regeneration is unknown. Therefore, to identify principles of regeneration that are conserved among bilaterians, we investigated the role of Hippo signaling in the limb bud regeneration of an anuran amphibian, Xenopus laevis. We found that a transcription factor, Yap1, an important downstream effector of Hippo signaling, is upregulated in the regenerating limb bud. To evaluate Yap1׳s function in limb bud regeneration, we made transgenic animals that expressed a dominant-negative form of Yap under a heat-shock promoter. Overexpression of a dominant-negative form of Yap in tadpoles reduced cell proliferation, induced ectopic apoptosis, perturbed the expression domains of limb-patterning genes including hoxa13, hoxa11, and shh in the regenerating limb bud. Transient expression of a dominant-negative Yap in transgenic tadpoles also caused limb bud regeneration defects, and reduced intercalary regeneration.These results indicate that Yap1 has a crucial role in controlling the limb regenerative capacity in Xenopus, and suggest that the involvement of Hippo signaling in regeneration is conserved between vertebrates and invertebrates. This finding provides molecular evidence that common principles underlie regeneration across phyla, and may contribute to the development of new therapies in regenerative medicine.

Biolistic transformation ofcry 1Ac gene in eggplant (Solanum melongenaL.)

Among different explants, cotyledon showed highest regeneration and number of buds per explant on all media combinations. However, highest regeneration potential was observed on MS fortified with 3.0 mgl−1 BAP (80.36%), but number of buds per explant were highest (21.76) on MS media supplemented with 2.5 mgl−1 BAP+ 1.0 mgl−1 Kin. Plant regeneration of cotyledons was completely checked with a selection pressure of 15ppm to 25ppm hygromycin for untranformed explants. But the selection efficacy of transformed tissue was 19.56% with 20ppm and 66.66% with 15 ppm hygromycin. GUS gene was expressed in 76.92% bombarded explants with 2 to 7 GUS spots per explant confirming the presence of transformed tissue. GUS gene was also visualized in 66.66% regenerated shoots from bombarded tissue. PCR analysis of putative transgenic eggplant plants with the specific primers confirmed the presence of Cry1Ac gene with 1.2 KB product size (falling between 1KB and 2KB bands of 10KB DNA ladder) in two of the four plants.

Axillary Bud Propagation and Regeneration from Stem Segment Explants in Calophyllum inophyllum

Axillary Bud Propagation and Regeneration from Stem Segment Explants in <i>Calophyllum inophyllum</i>

Adventitious bud regeneration from cotyledonary node, hypocotyl, cotyledon and euphylla of various genotypes of sunflower (Helianthus annuusL.)

Isolated culture of sunflower is significantly important in seeking new species of sunflowers with plenty of essential oil by modern biotechnologies. Thus, the callus development and adventitious bud regeneration from various explants of sunflower were studied by in vitro tissue cultures. It was found that the callus were readily formed on six genotype explants cultured in media with IAA and 6-BA, but adventitious bud regeneration from these callus was relatively weak. The adventitious bud regeneration inductivities of four tissue explants from different genotype sunflowers were ordered as followed: cotyledonary nodes > hypocotyls > cotyledons > euphyllas. The desired concentrations of 6-BA inducing adventitious bud regeneration were at 1.2–1.8 mg/l for cotyledonary nodes, 1.8 mg/l for hypocotyls and 0.6 mg/l for cotyledons, respectively. But no adventitious buds were differentiated from euphyllas only by 6-BA. The best medium for organogenesis was composed of MS+0.03 mg/l IAA+1.2–1.5 mg/6-BA by which euphyllas also were induced to regenerate adventitious buds with an averagely 20.83% inductivity. Among several genotype sunflowers, PR29 had the highest regeneration frequency and the regenerated plantlet has been successfully cultured.

Response of cotyledon explants of Capsicum annuum L. cv. kujawianka to chosen plant growth regulators in in vitro culture

Shoot buds originated directly on cotyledon explants of &lt;em&gt;Capsicum annuum&lt;/em&gt; L. cv. Kujawianka, when Linsmaier and Skoog medium was enriched with BAP (2 mg/l). Kinetin (2 mg/l) or kinetin with IAA (1 mg/l + 1 mg/l) induced indirect shoot buds regeneration from callus. Rooting was obtained with explants cultivated on a medium containing NAA (0,5 mg/l). Occurrence of the early stages of differentiation was proved at the histological level.

Rapid In Vitro Plant Regeneration of Black Gram (Vigna mungo L. Hepper) Var. Sarala, an Important Legume Crop

Efficient in vitro regeneration of black gram (Vigna mungo L. Hepper) var. Sarala was achieved through organogenesis using cotyledonary explants excised from 4 days old seedlings on MS medium supplemented with 2.0 mg/l BAP. Organogenic calli were developed from cotyledonary tissues within 4–6 weeks of culture on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BAP) along with 2.0 mg/l 1-napthaleneacetic acid (NAA). Shoot bud regeneration was achieved on MS medium supplemented with 2.0 mg/l BAP within 3–4 weeks of subculture. The number of shoots per culture varied from 1.12 to 8.75 in different growth media. The cultures incubated initially on dark photoperiod for 2 weeks and subsequently transferred to 16 h photoperiod showed higher number of shoot bud regeneration. The proliferated shoots were further sub-cultured on similar medium for higher rate of shoot bud regeneration. The elongated shoots were rooted on ½ strength MS medium fortified with 0.1–0.5 mg/l NAA or indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) with 2 % (w/v) sucrose within 2–3 weeks of culture. The higher percentage of rooting was obtained on 0.1–0.25 mg/l NAA as compared with IBA or IAA. The rooted plantlets were transferred to soil mixture (soil: sand: vermi-compost, 1:1:1 ratio) and kept in the greenhouse with 85 % humidity. The regenerated plantlets were successfully grown with 75 % survival rate. This protocol can be used for genetic improvement of black gram.

Comparative analysis of direct plant regeneration from immature leaf whorl and floral explants for three elite US sugarcane (Saccharum spp. hybrids) genotypes

Sugarcane (Saccharum spp. hybrids) is an important commodity field crop in tropical and subtropical countries providing sugar and biofuel feedstock and occupying a critical and strategic position in the global economy. This study was conducted to evaluate, compare, and optimize a rapid direct regeneration tissue culture system from immature leaf whorl and pre-emergent floral explants for three elite US sugarcane genotypes: CP84-1198, CP88-1762, and CP89-2143. Direct regeneration of adventitious shoot buds from the immature leaf roll explants and subsequent elongation and rooting of shoot buds was successfully obtained on modified Murashige and Skoog salt medium supplemented with 5 mg l–1 α-naphthaleneacetic acid and 0.5 mg l−1 kinetin. Significant genotype-specific differences in the morphogenetic potential of leaf roll explants were discernible with the explant developmental stage (explant position along the leaf roll axis) and orientation during in vitro culture. The highest number of shoots was regenerated from CP88-1762, followed by CP89-2143 and CP84-1198 from explants closest to the meristem that were oriented horizontally (CP88-1762) or vertically (CP89-2143 and CP84-1198) on the culture medium. Immature inflorescence-derived explants from all three genotypes when cultured on the above medium for 2 wk rapidly produced shoots, followed by rooting on medium supplemented with 4 mg l−1 indole-3-butyric acid. The regeneration protocols yielded robust rooted plantlets from immature leaf roll explants within 4 to 6 wk, which were readily acclimatized under greenhouse conditions.

Development of a seedling clone with high regeneration capacity and susceptibility to Agrobacterium in apple

Agrobacterium-mediated transformation has been the preferred system for transforming apples. Although transgenic lines have been obtained form a number of genotypes, the transformation efficiency was not high. Apple is highly heterozygous genetically, so its seedling populations segregate heavily. We speculate that a genotype with high regeneration capacity and sensitive to Agrobacterium could be screened from the seedling population of apple. In order to verify this hypothesis, we developed in vitro seedling clones of apple (Malus×domestica) and compared their regeneration capacity and susceptibility to Agrobacterium tumefaciens in this study. Royal Gala, a genotype had been widely used in apple transformation, was selected as the source of seeds. First, 10 clones with higher multiplication capacity and normal leaf shape and smooth leaf surface, named GL-1 to GL-10, were screened out from 100 in vitro seedling clones. The capacity of adventitious bud regeneration from leaf segments were compared among the ten clones, and the result showed that GL-3 exhibited extremely higher adventitious regeneration capacity. GL-3 was also compared with its parent on the regeneration capacity, and the data showed that the number of buds per explant of GL-3 was 2.6 fold higher than that of Royal Gala. The susceptibility of GL-3 to Agrobacterium was compared with Royal Gala by monitoring the transient expression of GUS in leaf segments. For GL-3, the percentage of explants with large GUS-positive patches was 46.7±1.1%, which was obviously higher than that (25.1±7.2%) of Royal Gala. After the leaf segments of GL-3 were transformed with A. tumefaciens strain EHA105 carrying the AtmiR156a antisense gene, the percentage of explants with kanamycin-resistant buds reached 27.5%. In conclusion, a genotype with high regeneration capacity and sensitive to Agrobacterium was screened out from the seedling population of apple, and this strategy could be applied in other perennial woody plants.

Evaluation of Genetic Homogeneity in Tissue Culture Regenerates of Jatropha curcas L. using Flow Cytometer and DNA-based Molecular Markers

The present investigation aimed to evaluate the reliability of in vitro propagation methods for elite genotypes of Jatropha curcas L., that maintain genetic integrity of tissue culture (TC) regenerates among two regeneration systems developed through direct shoot bud regeneration using nodal/apical shoot segments (protocol-A) and in vitro-derived leaves (protocol-B) as explants. Random amplified polymorphic DNA (RAPD), intersimple sequence repeat (ISSR), simple sequence repeat (SSR) molecular markers, and flow cytometery (FCM) were employed to evaluate genetic homogeneity in TC-regenerates at different passages of subcultures. RAPD markers showed genetic homogeneity in fifth-generation TC-regenerates of both protocols. ISSR markers showed genetic stability of leaf regenerates (protocol-B) at 10th generation. FCM analysis of TC-regenerates at 10th generation in protocol-B and at 20th generation in both protocols, showed stability of ploidy level. SSR assessment of TC-regenerates at 20th generation in both protocols confirmed genetic homogeneity. The results confirmed the genetic stability of the TC-regenerates and demonstrated the reliability of the regeneration systems developed so far using explants of two different origins, for large-scale multiplication of elite genotypes of Jatropha.

Plant regeneration protocol of Andrographis paniculata (Burm. f.) - an important medicinal plant

Rapid direct plant regeneration of Andrographis paniculata was achieved from leaf and stem explants on Murashige and Skoog (MS) basal medium supplemented with 1.5 to 3.0 mg/l 6-benzyladenine (BA), 50 mg/l adenine sulfate (Ads) and 3% (m/v) sucrose. Inclusion of 1.0 mg/l 1-naphthalene acetic acid (NAA) in the culture medium along with BA + Ads promoted a higher rate of shoot bud regeneration. Maximum mean number of shoot bud per explant (28.6) was achieved on the MS medium supplemented with 3.0 mg/l BA, 50 mg/l Ads and 1.0 mg/l NAA after six weeks of culture. The percent of regeneration varied depending on the culture medium. The elongated shoots were rooted within 9 to 11 days on ½ strength MS medium supplemented with 0.5 mg/l indole-3-butyric acid (IBA) or 1-naphthaleneacetic (NAA) acid with 2% sucrose. Maximum percentage of rooting (76.24%) was obtained on medium having 0.5 mg/l IBA and 2% sucrose. Basal region of the micro-shoots became callusing when transferred to higher concentrations of either IBA or NAA. The rooted plantlets were survived in the greenhouse. The in vitro raised plantlets were grown normally under soil condition. This result will facilitate the conservation and propagation of the important medicinal plant. Keywords : In vitro , shoot bud regeneration, growth regulators, medicinal plants. African Journal of Biotechnology Vol. 12(39), pp. 5738-5742

Regeneración de plantas a partir de cultivos de callos de &lt;i&gt;Vitex trifolia&lt;/i&gt; (Lamiales: Lamiaceae): una planta medicinal potencial

Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supplemented Murashige & Skoog medium, and incubated at 25 +/-2 degrees C under a light intensity of 61 micromol/m2s from cool white fluorescent lamps and a 16 h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10 microM BAP in combination with 0.54 microMNAA. Addition of 135.74-271.50 microM adenine sulphate (Ads) and 0.72-1.44 microM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10 microM BAP in combination with 0.54 microM NAA, 271.50 microM Ads and 1.44 microM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23 microM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

A reproducible and high frequency plant regeneration from mature axillary node explants of Gymnema sylvestre (Gurmur)—An important antidiabetic endangered medicinal plant

Gymnema sylvestre is an important medicinal plant used in different systems of medicine as a remedy for the treatment of diabetes. The present study describes an efficient and rapid protocol for large scale in vitro plant regeneration from mature axillary node explants of G. sylvestre. The axillary node explants were cultured on MS medium supplemented with different concentrations of BAP and KIN (0.5–3.0mg/l) for shoot bud induction. In order to enhance the shoot bud multiplication, regenerated shoot buds were further subcultured onto MS medium fortified with different concentrations of BAP (0.5–3.0mg/l) in combination with 0.5mg/l of NAA/IBA/IAA/KIN. The highest frequency (84.22%) of multiple shoot bud regeneration with maximum number of shoots (14.20shoots/explant) was noticed on MS medium supplemented with 1.0mg/l BAP and 0.5mg/l KIN combination. In another experiment, in vitro derived shoot buds were cultured on different concentrations of GA3 (0.5–2.0mg/l) and various concentrations of BAP (0.5–3.0mg/l) in combination with KIN (0.5mg/l) and GA3 (1.0mg/l) for shoot bud multiplications as well as elongation. For large scale plant production, in vitro derived axillary buds were cultured on MS medium fortified with BAP (1.0mg/l)+KIN (0.5mg/l)+GA3 (1.0mg/l) combination, in which about 418.72shoots/explant were obtained after five subcultures on the same media composition. Elongated shoots (>2cm) were dissected out from the in vitro proliferated shoot clumps and were cultured on half-strength MS medium containing different concentrations of various auxins (IAA, IBA and NAA) (0.5–2.0mg/l) for root induction. Highest frequency of rooting (78%) was noticed on half-strength MS medium augmented with 2.0mg/l IBA. The rooted plantlets were successfully transferred into plastic cups containing sand and soil in the ratio of 1:2 and subsequently they were established in the greenhouse. The present in vitro regeneration protocol would facilitate an alternative method for fast and large scale propagation of this endangered antidiabetic medicinal plant.

Shoot regeneration from various explants of horse chestnut (Aesculus hippocastanum L.)

A protocol is described for the adventitious shoot bud regeneration from leaf and stem explants in Aesculus hippocastanum. Effect of different media (Heller, WPM, Rugini and DKW) and growth regulators (BA, TDZ and NAA) combinations on shoot organogenesis were examined. Culture medium content had a significant effect on shoot regeneration in Aesculus. The highest shoot number and length was achieved on Rugini and WPM medium. Adventitious shoot formation was observed in all types of explants. Regeneration frequency and shoot number depended on explant type and growth regulators. The highest regeneration (100%) and the highest shoot bud number (9.7) were recorded in stem explants on WPM medium with 8.9μM BA+0.5μM NAA. Generally, TDZ was less effective on shoot regeneration in stem and leaf explants compared to BA. The addition of NAA to the culture medium improved shoot regeneration from stem discs. Rooting of elongated shoots was significantly increased (68.1%) on full-strength WPM medium supplemented with 2.7μM NAA. Rooted plantlets were acclimatized successfully under greenhouse conditions.

Development of an efficient regeneration and Agrobacterium-mediated transformation system in crab apple (Malus micromalus) using cotyledons as explants

Apple has become a model species for Rosaceae genetic and genomic research, but it is difficult to obtain transgenic apple plants by Agrobacterium-mediated transformation using in vitro leaves as explants. In this study, we developed an efficient regeneration and Agrobacterium-mediated transformation system for crab apple (Malus micromalus) using cotyledons as explants. The proximal cotyledons of M. micromalus, excised from seedlings that emerged from mature embryos cultured for 10–14 d in vitro, were suitable as explants for regeneration and Agrobacterium-mediated transformation. Cotyledon explants were cocultivated for 3 d with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA2301 on regeneration medium. Kanamycin-resistant buds were produced on cotyledon explants cultured on selective regeneration medium containing 20 mg/L kanamycin. Acetosyringone supplemented in the Agrobacterium suspension or in the cocultivation medium slightly enhanced the regeneration of kanamycin-resistant buds. The maximum percentage of explants with kanamycin-resistant buds was 11.7%. The putative transformed plants were confirmed by histochemical analysis of β-glucuronidase activity and the polymerase chain reaction amplification of the neomycin phosphotransferase II gene. This transformation system also enables recovery of nontransformed isogenic controls developed from embryo buds and is therefore suitable for functional genomics studies in apple.

Divergent requirements for fibroblast growth factor signaling in zebrafish maxillary barbel and caudal fin regeneration

The zebrafish maxillary barbel is an integumentary organ containing skin, glands, pigment cells, taste buds, nerves, and endothelial vessels. The maxillary barbel can regenerate (LeClair & Topczewski 2010); however, little is known about its molecular regulation. We have studied fibroblast growth factor (FGF) pathway molecules during barbel regeneration, comparing this system to a well-known regenerating appendage, the zebrafish caudal fin. Multiple FGF ligands (fgf20a, fgf24), receptors (fgfr1-4) and downstream targets (pea3, il17d) are expressed in normal and regenerating barbel tissue, confirming FGF activation. To test if specific FGF pathways were required for barbel regeneration, we performed simultaneous barbel and caudal fin amputations in two temperature-dependent zebrafish lines. Zebrafish homozygous for a point mutation in fgf20a, a factor essential for caudal fin blastema formation, regrew maxillary barbels normally, indicating that the requirement for this ligand is appendage-specific. Global overexpression of a dominant negative FGF receptor, Tg(hsp70l:dn-fgfr1:EGFP)(pd1) completely blocked fin outgrowth but only partially inhibited barbel outgrowth, suggesting reduced requirements for FGFs in barbel tissue. Maxillary barbels expressing dn-fgfr1 regenerated peripheral nerves, dermal connective tissue, endothelial tubes, and a glandular epithelium; in contrast to a recent report in which dn-fgfr1 overexpression blocks pharyngeal taste bud formation in zebrafish larvae (Kapsimali et al. 2011), we observed robust formation of calretinin-positive tastebuds. These are the first experiments to explore the molecular mechanisms of maxillary barbel regeneration. Our results suggest heterogeneous requirements for FGF signaling in the regeneration of different zebrafish appendages (caudal fin versus maxillary barbel) and taste buds of different embryonic origin (pharyngeal endoderm versus barbel ectoderm).