Shoot Apex Culture Research Articles

Assessment of in vitro growth of apical stem sections and adventitious organogenesis to evaluate salinity tolerance in cultivated tomato

The possible use of in vitro shoot morphogenesis and shoot apex culture to evaluate salt tolerance in cultivated tomato (Lycopersicon esculentum Mill.) has been analyzed, using two cultivars with similar salt tolerance, Pera and Hellfrucht fruhstamm (HF). The effect of salt on shoot regeneration was studied by culturing leaf explants on media supplemented with 0, 43, 86, 129 and 172 mM NaCl. The presence of NaCl in the regeneration media at 86 mM strongly inhibited shoot regeneration in the cultivar HF, but not in Pera. However, the substitution of NaCl by mannitol, maintaining the same water potential in the culture media, decreased the regeneration percentage in Pera but did not affect HF. Shoot apices of both cultivars were also subcultured at 6-week intervals, for 4 subcultures, at the same NaCl concentrations as used in the previous experiment, and the shoot growth, leaf and root number, rooted shoot and shoot necrosis were recorded at the end of each subculture. Root formation was the parameter most affected by salt in both cultivars, Pera being more sensitive than HF. The substitution of NaCl by mannitol significantly increased the percentage of rooted shoots in Pera after four subcultures, and slightly decreased this percentage in HF. Shoot necrosis was only observed in the last subculture at NaCl higher than 86 mM, the percentage of necrotic shoots being higher in Pera than in HF (75% and 45%, respectively). The lack of agreement between the results obtained with the in vitro tests, e.g., adventitious shoot formation and growth of apical stem sections, suggests that this approach may not be a reliable tool to evaluate salt tolerance in cultivated tomato.

Restoration of Juvenility in Maize Shoots by Meristem Culture

Shoot-apex culture in maize (Zea mays L.), which results in the formation of an entire new shoot by the cultured meristem, was used to examine phase change. In phase change, patterns of differentiation of leaves, axillary buds, and nodes change in a coordinated manner as the plant matures from a juvenile to an adult. The culturing of meristems from adult-phase plants restored the ability of the plant to form leaves with juvenile traits, including having epicuticular wax, a thin cuticle, violet staining with toluidine blue, and round epidermal cells. Although shoots from cultured apices had a few extra nodes compared to seed-derived plants, the upper, adult-like portion of the culture-derived shoots were identical to the adult region of normal maize plants in such traits as position of ear shoot, size and shape of leaves, and the presence of trichomes on leaves. These results support the conclusion that phase-specific patterns of differentiation in the shoot are the result of signaling throughout the plant and not the age of the meristem.

Evaluation of salt tolerance in cultivated and wild tomato species through in vitro shoot apex culture

The possibility of using in vitro shoot apex culture to evaluate salt tolerance of cultivated (Lycopersicon esculentum Mill.) and wild (Lycopersicon pennellii (Correll) D'Arcy) tomato species was determined and related to the response obtained by callus culture. Both apices and calluses were grown on media supplemented with 0, 35, 70, 105, 140, 175 and 210 mM NaCl, and growth and physiological traits were determined. Most apices of L. esculentum did not develop roots from low NaCl levels, whereas the apices of L. pennellii were able to develop roots at the different salt levels. This different degree of salt tolerance between L. esculentum and L. pennellii was not, however, clearly shown on the basis of the shoot growth of the plantlets. The callus response was similar to that shown by the rooting parameters, as callus growth in response to increased salinity was much greater in L. pennellii than in the tomato cultivar. K+decreased more and proline accumulated less with salinity in shoots of L. esculentum compared to L. pennellii, whereas the opposite response was obtained in calluses. The results obtained in this study suggest that rooting parameters are the most useful traits for rapid evaluation and screening of tomato species and segregating populations through in vitro shoot apex culture.

Adaptation of Cotton Shoot Apex Culture to Agrobacterium-Mediated Transformation

A protocol is presented for rapid genotype-independent transformation and regeneration of cotton (Gossypium spp.) from shoots isolated from germinating seedlings. Isolated shoots are inoculated with a super-virulent strain of Agrobacterium tumefaciens, subjected to a mild antibiotic selection, and directly regenerated as shoots in vitro. Shoots do not dedifferentiate and mutation rates are low. Rooted shoots can be obtained within 6–10 weeks of isolation and inoculation depending on the cotton cultivar.

The Effects of Growth Regulators on Shoot Propagation and Rooting of Vitis Following in Vitro Axillary Bud and Shoot Apex Culture

Axillary buds of `Valiant' grapevine (Vitis spp.) grown in vitro were transferred onto Murashige and Skoog (MS) medium supplemented with different cytokinin and auxin combinations and concentrations. It was found that culture medium caused statistically important differences in number of nodes, number of fully expanded leaves, number of multiple shoots, number of roots, and length of shoots. MS medium supplemented with 1.0 mg BA/liter in combination with 0.01 mg NAA/L was found to be the best medium for shoot growth and callus production. MS medium supplemented with the combination of 0.5 mg BA/L and 0.01 mg NAA/L was the best medium for explant rooting. The medium containing BA and NAA encouraged better shoot growth than those containing BA alone. When the concentration of BA in the medium was increased, multiple shoot proliferation and teratological structures of explants increased, but the number of small leaves and length of internode decreased. Axillary bud culture led to better shoot growth than was found for shoot apex culture. The presence of leaves positively affected shoot growth from axillary buds. Also placing the axillary buds horizontally onto the medium gave better shoot proliferation and growth than placing them vertically.

Regulation of extent of vegetative development of the maize shoot meristem

In maize plants (Zea mays L.), the extent of vegetative development in the shoot is precisely regulated such that the apical meristem produces a predictable number of leaves before converting to tassel development. In previous experiments using shoot apex culture, we showed that the developmental program that limits vegetative development in maize is not intrinsic to the shoot apical meristem. Rather, the meristem receives information from elsewhere in the plant and responds by either continuing leaf initiation or becoming determined for determinate growth and forming an inflorescence, the tassel. Here we examine leaf primordia as potential sources for that information using shoot apex culture. Our results show that the presence of the four to six youngest leaf primordia on the shoot apex is sufficient to provide such information. The ability to reset shoot development by meristem culture also allows us to examine the basis for expression of a specific phenotype at a particular developmental stage. We found that the mutation hcf106, which is typically expressed only during seedling stages, is not re‐expressed when the shoot morphogically has regained a juvenile phase.

In vitro Flowering and Fruiting of Strawberry through Shoot Apex Culture

A method of inducing in vitro flowering and fruiting of strawberry plants obtained by shoot apex culture was investigated. When strawberry plants were cultured in the flask (300 ml), the crown did not enlarge over 3 mm diameter, whereas when cultured in a culture-bottle (100mm × 110mm × 100mm), the crown enlarged to 5 mm diameter.A June-bearing type strawberry, 'Toyonoka', and an ever-bearing type, 'Summerberry' with 3 mm crown diameters did not differentiate flower buds, whereas those with 5 mm crown diameters were induced to flowere.The percentages of flower buds differentiated by 'Toyonoka' and 'Summerberry' crowns were 22.2 % and 84.2 %, respectively, whereas only 11.1 % and 36.8 %, respectively, flowered.Thus, culture-bottle system enabled in vitro flowering of strawberry. Using this system, fruits were obtained by artificial pollination.

イチゴの茎頂培養における幼植物体の生長におよぼすランナーの前処理と低温貯蔵の影響

イチゴの茎頂培養における幼植物体の生長におよぼすランナーの前処理と低温貯蔵の影響

A high sensitivity RT-PCR assay for the diagnosis of grapevine viroids in field and tissue culture samples.

An RNA extraction procedure, modified from published methods, and a high sensitivity reverse transcription-polymerase chain reaction (RT-PCR) assay have been developed for the detection of the five viroids in grapevines. All five viroids have been found in the 10 different grape varieties tested so far. This assay, specially optimised for viroids in low copy number by careful selection of DNA primers, has been used in conjunction with dot blot hybridization assay for the study of viroids in vines regenerated by shoot apical meristem culture (SAMC) and fragmented shoot apex culture (FSAC). The data indicate a differential reduction of viroids, rather than viroid elimination, in the regenerated vines. Transmission of viroids via grape seeds was also observed.

Influence of genotype and growth regulators on morphogenetic processes in carnation shoot apex cultures

Shoot tip explants from eight carnation cultivars were grown on Murashige and Skoog medium with or without growth regulators (μM): α-naphtaleneacetic acid (0.5) and kinetin (KIN) (2.5), maleic hydrazide (MH) (0.5) and KIN (2.5) or MH (0.5) and 6-benzylaminopurine (2.5). Most of the explants developed into shoots which formed a callus and/or roots at the basal region. Some of the explants developed flower buds or anomalous flowers. For all the media tested, significant differences among genotypes in shoot formation and growth efficiency, as well as in the rate of abnormality, rooting and callusing of the shoots were observed. The morphogenetic responses were also affected by the growth regulators. But shoot tip explants from different cultivars reacted to the modifying effects of growth regulators differently.

In vitro propagation of taro, with spermine, arginine and ornithine

Regeneration of plantlets from shoot apex-derived callus and "calloid" cultures of a local taro [Colocasia esculenta var. antiquorum cv. Keladi Birah] cultivar, was expedited by treatment with high levels of spermine. The total time taken, from culture of primary shoot apices on modified Linsmaier and Skoog medium supplemented with trichlorophenoxyacetic acid and kinetin, to complete plantlet regeneration, was reduced by 2-16 weeks, when the callus and "calloid" cultures were treated with 0.01, 0.1 and 1 mM spermine. Furthermore, the number of plantlets produced per gram callus increased from 25 to 55. On media supplemented with arginine and ornithine, no callus was initiated from expiants and no plantlets differentiated from pre-established callus.

Induction of Multiple shoots by Shoot Apex Culture in Magnolia obovata Thunb.

Shoot apex of Magnolia obovata Thunb. was cultured on the modified Murashige and Skoog's (MS) medium supplemented with the combinations of IBA and BAP or NAA and BAP in various concentrations. Multiple shoots were induced successfully on the half strength MS reducing nitrate to one fourth (1/4N-1/2MS) medium containing 4μM IBA and 5μM BAP after 40 days of culture. The multiple shoots obtained were divided into small nodal explants and further subcultured on fresh medium. Many axillary buds were induced from the explant after 20 days of culture, and they succeedingly grew actively and developed multiple shoots. Culturing the axillary bud together with the stem tissue was effective for facilitating the induction of multiple shoots.

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.

Evaluation of biological indexing and dsRNA analysis in grapevine virus elimination

SummaryFragmented shoot apex culture (FSAC) was used to eliminate some diseases caused by viruses and virus‐like agents from 24 varieties of imported grapevines. Each cultivar was examined by biological indexing before and after FSAC. Graft indexing revealed that leaf roll, stem pitting and yellow speckle diseases were common before FSAC. A correlation was observed between the incidence of leafroll disease and the presence of specific dsRNA species which were removed after FSAC. One of these species, an RNA of about one kilobase pair was associated with low yielding Sultana clones. Both biological indexing and dsRNA assay indicated no recurrence of the leafroll disease in material regenerated by FSAC even after 10 years in the field. It is concluded that dsRNA assay may be used as a faster and less expensive method than biological indexing in assessing the success of leafroll elimination by FSAC. The test also provides some information on the genome size of the viruses associated with leafroll disease. Graft indexing indicated that yellow speckle disease was resistant to elimination by FSAC, while stem pitting was removed from some of the vines and the grapevine fleck disease was eliminated from most sources.

Regeneration of Triticum aestivum apical explants after microinjection of germ line progenitor cells with DNA

A highly efficient method of regenerating fertile, phenotypically normal plants from shoot apex cultures of T. aestivum was developed. The hypodermal layer (L2) of the vegetative apex containing germ line precursor cells could be located with bright field microscopy and targeted for microinjection. Fluorescently labelled dextrans were used as markers to develop a microinjection procedure which did not disrupt nuclear or cytoplasmic structure. This procedure was used to inject plasmid DNA into L2 cells. Capillary microinjection did not shear the plasmid DNA and delivery of DNA was confirmed by polymerase chain reaction analysis of DNA isolated from injected apices. The significance of these findings in relation to the development of cereal transformation systems will be discussed.

Resistant Plants to Alternaria alternata Strawberry Pathotype Selected from Calliclones of Strawberry Cultivar Morioka-16 and Their Characteristics

To breed an improved 'Morioka-16' which is resistant to Alternaria black spot, caused by Alternaria alternate strawberry pathotype, culture-caused mutagenesis was used. Calliclones induced by shoot apex culture of cv. Morioka-16 were inoculated with the pathogen by three screening methods.Using one of these methods, three plants resistant to the disease were successfully selected from 1196 calliclones which had been inoculated with the pathogen.These three mutants were named 'M16-AR 1', 'M16-AR 2' and 'M16-AR 3' and their plant and fruit characters were examined. No differences between cv. Morioka-16 and the three mutants were found in terms of plant height, leaf number, flowering time, harvesting time, yield, and fruit characters.

Identification of multiple stages in the conversion of maize meristems from vegetative to floral development

ABSTRACT Vegetative growth in most lines of maize is terminated at a predictable stage in development by the conversion of the shoot apical meristem into an inflorescence, the tassel. The conversion from vegetative to floral development is under developmental control, the basis of which is obscure. We have assayed the developmental potential of the shoot apical meristem in order to identify the stage at which it is determined to form a tassel. We show, using shoot apex culture, that meristems are not determined to form a tassel until after all vegetative nodes have been initiated. We also show that floral determination is a separate, later event in the development of a maize inflorescence. Intermediate stages can be distinguished in which the meristem is determinate and has the phyllotaxis of a tassel when cultured but develops vegetative shoots from buds that normally give rise to sets of florets.

Paulownia witches' broomfree plantlets regenerated from the axillary buds of infected Paulownia tomentosa STEUD mature trees by shoot apex culture

Paulownia witches' broomfree plantlets regenerated from the axillary buds of infected Paulownia tomentosa STEUD mature trees by shoot apex culture

Somatic embryogenesis and plant regeneration from stem, leaf, and shoot apex of melon (Cucumis melo L.).

Somatic embryogenesis and plant regeneration were investigated in stem, leaf, and shoot apex culture of melon (Cucumis melo L.). Stems of 3 varieties which had grown in a greenhouse 7-12 weeks after transplanting were cultured on Murashige and Skoog (MS) medium supplemented with 2, 4-dichlorophenoxyacetic acid (2, 4-D), 2-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BA) and casein hydrolysate (CH). Somatic embryos developed from the explants in all tested varieties, but the appropriate concentrations of growth regulators and CH for somatic embryogenesis were different among the varieties. In leaf and shoot apex culture, the leaves and shoot apexes from aseptic plants of 2 and 11 varieties, respectively, were cultured on MS medium supplemented with 2, 4-D, BA, and sucrose. Somatic embryos developed from the leaf in both tested varieties and from shoot apex in ten tested varieties. Sucrose concentration influenced somatic embryogenesis in these cultures. Plant regeneration was obtained by subculturing somatic embryos derived from stem, leaf, and shoot apex on hormone-free MS medium.

Propagation of Oenothera erythrosepala Borbas by shoot apex culture.

Shoot apices of Oenothera erythrosepala were cultured on MS medium supplemented with various concentrations and combinations of naphthaleneaceticacid (NAA) and 6-benzylaminopurine (BA). Effect of hormonal concentration on number of newly induced shoots per shoot apex placed was evident. The media containing 0.2mg/l NAA 0.2mg/l BA and/or 0.2mg/l NAA 2.0mg/l BA were suitable for shoot multiplication and 2.0mg/l NAA for rooting. There was a little difference in root formation and root hair development according to the gelling agents used, but the survival rate was not affected by them.