Adventitious Shoot Regeneration in Carnation (Dianthus caryophyllus) from Axillary Bud Explants

Adventitious shoots were regenerated from axillary bud explantsof 15 carnation cultivars. The use of leaf and stem explantswas not successful, largely due to explant senescence in thepresence of benzyladenine, kinetin and, to a lesser extent,zeatin. For axillary bud explants, a suitable optimum adventitiousregeneration medium contained Murashige and Skoog basal mediumsolidified with Gelrite and supplemented with 15 µm benzyladenineand 0.5 µM a-napthaleneacetic acid. Adventitious primordiaarose from the cut basal end of bud explants erupting as individualshoots after 2–3 weeks incubation. The axillary bud sizeand the time between subcultures of source material influencedthe production of adventitious shoots. Transfer of regeneratedshoots onto a medium solidified with agar minimized visiblesigns of vitrification. Regenerated shoots could be easily rooted,transferred to glasshouse conditions and grown to flowering. Vitrification, tissue culture, cut flowers, Dianthus caryophyllus L., carnation, cytokinins, explant     

Cytokinin as an Inducer of Vitrification in Melon

Development of vitrification has been followed in melon shoottips cultured on solid and liquid media which contained growthfactors in various concentrations. Cytokinin was found to bethe primary inducer, without which vitrification did not occurin spite of the presence in the culture of other accepted inducers.Spontaneous vitrification appeared on non-inductive media butin a low percentage. Vitrification differed in appearance andfrequency, depending on the kind of medium. On solid mediumvitrification gradually increased with time, whereas on liquidmedium it was an ‘all-or-nothing’ effect. On solidmedium, all the vitrified plantlets turned into callus aftertwo or three subcultures. It is suggested that vitrificationis the bud's reaction to an excess of cytokinin. Cucumis melo, growth factors, abnormal plant growth, vitrification     

Mass Propagation of Eucalyptus camaldulensis in a Scaled-up Vessel Under In Vitro Photoautotrophic Condition

A scaled-up culture vessel was designed for the large-scalephotoautotrophic micropropagation of chlorophyllous plants.The culture vessel (volume 20 l) contained a plug cell traywith 448 plantlets, and had a forced ventilation system to supplyCO₂-enriched air. A nutrient-reservoir was connected to theculture vessel from which nutrient solution was circulated tothe culture vessel every 24 h. Nodal leafy cuttings of Eucalyptuscamaldulensis L. were cultured photoautotrophically in thissystem without sugar in the nutrient medium, but with an enrichedCO₂concentration and a high photosynthetic photon flux. Thegrowth and the net photosynthetic rate of the in vitro grownplantlets and the survival percentage of the plantlets aftertransplanting to ex vitro conditions were compared with thoseof plantlets grown photoautotrophically under natural ventilationin conventional small culture vessels (Magenta-type vessels;volume 0.4 l). Fresh and dry masses and net photosynthetic ratewere significantly higher in plantlets grown in the scaled-upvessel compared to plantlets grown in the conventional smallvessels (control). The environmental conditions created in thisscaled-up vessel (with forced ventilation) also facilitatedacclimatizationin vitro . Importantly, after transplanting tothe ex vitro condition, plantlets grew well without any specializedexvitro acclimatization treatment. Copyright 2000 Annals of BotanyCompany CO₂enrichment, Eucalyptus camaldulensis L., ex vitro, forced ventilation, natural ventilation, photoautotrophic, scaled-up vessel, survival percentage     

'Vitrified' Dianthus--Teratomata in vitro due to Growth Factor Imbalance

Cultured shoot tips of Dianthus caryophyllus often develop asabnormal, ‘vitrified’ plants that have the characteristicsof teratomata: stable, unlimited development with fasciatedshoot apices, a bush habit, reduced stem elongation and succulentleaves. High concentrations of NAA in the culture medium increasedand benzylaminopurine decreased the proportion of shoot-tipsthat developed as abnormal plants. The requirements of abnormaland normal plants for continued development differed. Duringthe initial stages of development abnormal plants did not requireauxin and they were able to continue their growth in the absenceof roots. It is suggested that the abnormal plants are teratomata,similar to those induced by Crown Gall bacteria, and their inductionand abnormal traits could be due to imbalance of auxins andcytokinins. Dianthus caryophyllus L., carnation, abnormal plant growth, habituation, vitrification, teratoma (plant)     

Promoter Deletions are Essential for Transformation of Lettuce by the T-cytGene: The Phenotypes of Transgenic Plants

TheAgrobacteriumT-cytgene was transferred into lettuce,Latucasativa‘Saladin’ using a genotype-independent transformationprocedure employing a supervirulentAgrobacterium tumefaciensstraincarrying the binary vector pMOG23. Kanamycin-resistant shootswere initiated from inoculated explants only when sites weredeleted within the T-cytpromoter. In culture, kanamycin-resistantshoots exhibited several phenotypes, including gall production,reduced internode length (dwarfism) and vitrification togetherwith differences in rooting. Rooted, neomycin phosphotransferase(NPTII)-positive plants recovered from their abnormal phenotypeand self pollinated to produce viable seed, following transferto the glasshouse. Kanamycin-resistant lettuce plants exhibitedincreased cytokinin and chlorophyll contents compared to non-transformedplants, physiological features which could benefit long-termstorage of this leafy vegetable.Copyright 1999 Annals of BotanyCompany Agrobacterium-mediated transformation, cytokinins,Lactuca sativaL., pigmentation, T-cytexpression, lettuce.     

Growth of Carnation Meristems in vitro: Anatomical Structure of Abnormal Plantlets and the Effect of Agar Concentration in the Medium on their Formation

Carnation meristems cultured in vitro grow into shoots of threetypes: normal, translucent and succulent. The apical meristemof succulent shoots was of the mantle-core type and it lackedpro-vascular tissue. The leaf had large vacuolated mesophyllcells, fewer stomata (often plugged), and no plate meristem.A higher agar concentration in the medium increased the percentageof normal shoots developing. This supports other indicationsthat the water potential of the medium affects morphogenesis. carnation, Dianthus caryophyllus L, meristem culture, abnormal plantlets, shoot meristem     

Generation of Seakale (Crambe maritima L.) Plantlets by Tissue Culture

When placed in shaken liquid culture in MS medium, callus derivedfrom petioles of seakale (Crambe maritima L.) failed to fragmentto produce a cell suspension culture. In the absence of 2,4-D,growth was very good, and hollow, dark green, trumpet-shapedbodies were produced, but in the presence of 2,4-D, solid, palergreen pear-shaped bodies were formed. When grown on filter paperbridges with White's medium, a small proportion of both typesof bodies underwent slow, partly-controlled development culminating,after several months, in the formation of plantlets. Seakale, Crambe maritima L., in vitro propagation, callus culture     

Enhancement of Tuberization of Axillary Shoot Buds of Potato (Solarium tuberosum L.) Cultivars Cultured In Vitro

Factors controlling growth and tuberization of axillary budsin shoots of plantlets of potato (Solarium tuberosum L.) culturedin vitro were investigated. Correlative inhibition restrainedgrowth and tuberization of the axillary buds. Exposure of intactplantlets for various periods (4 to 48 h) to low (2 or 12C)or high (30 C) temperatures as comparedto 18C, did not alleviatecorrelative inhibition. Removal of the apical part of the shoot,the roots or both was generally ineffective Elevating sucroseconcentration from 30 to 80 g dm^–3 promoted tuberizationon axillary buds, and the cytokinin 6-(-dimethylallylamino)purine (2iP), alleviated correlative inhibition and enhancedtuberization in intact plantlets. In the whole plantlet mostof the tubers were formed on the basal nodes, however, oncecorrelative inhibition was eliminated by the dissection of theshoot to single node sections, tubers were formed on every axillarybud. The single most effective factor inducing tuberizationin single node sections was the growth retardant ancymidol,an inhibitor of giberellin biosynthesis. Key words: Potato, Solanum tuberosum L., in vitro tuberization, correlative inhibition     

Studies on Endogenous Cytokinins in Guava (Psidium guajava L.)

The natural occurrence of cytokinins was examined in seededand seedless fruits of guava (Psidium guajava L.). Five differentcytokinins were isolated and three of them were tentativelyidentified as zeatin, zeatin riboside and zeatin nucleotide.Quantitative differences in cytokinin content were observedin the two types of fruits. Psidium guajava L., guava, cytokinin     

The Carnation Succulent Plantlet -- A Stable Teratological Growth

Axillary buds of carnation (cv. Cerise Royalette) cultured invitro, frequently became ‘succulent’ plantlets,which proved to be a teratalogical stable type of growth. Agarconcentration (0.8–1.2 per cent) in the medium influencedthe type of development, and 0.05, 1 or 2 mg l^–1 of NAAin the medium did not change the results. The succulent plantletsdid not revert to normal growth when transferred to medium containingmore agar, which favoured normal plantlet development. Succulentexcised meristems developed mainly into succulent plantlets. A hypothesis is made that a rearrangement of the meristem occursin the first days of growth, the consequence of which is thesucculent plantlet, which is no longer influenced by agar concentrationin the medium. Carnation, Dianthus caryophyllus L. cv. Cerise Royalette, vegetative shoot meristem, agar effect, meristem organization     

Growth and development of carnation ‘Dreambyul’ plantlets in a temporary immersion system and comparisons with conventional solid culture methods

The aim of the current study was to compare the effects of the culture method—conventional solid medium culture and temporary immersion system (TIS)—on the growth and development of carnation ‘Dreambyul’ plantlets. At the same time, different immersion intervals and immersion durations of TIS culture were also tested to find the optimal setting for mass production of high-quality carnation plantlets in vitro. In the first experiment, the results showed that the shoot length, root length, and number of nodes of plantlets cultured in the TIS were highest when the immersion interval was 8 h. Compared with that of plantlets cultured in the conventional solid medium culture, the fresh weight of plantlets cultured in the TIS was at least 3 times greater. The greatest total chlorophyll content, stomata with normal shapes was observed for plantlets grown in the TIS with an 8-h immersion interval. The lowest H₂O₂ level was recorded in plantlets cultured with the 8-h immersion interval. In the second study, growth traits such as the shoot length, root length, and stem diameter, as well as the number of shoots and roots tended to increase with immersion durations, and reached their peaks when the immersion duration was 90 s. Excessive water accumulation in tissues and a higher incidence of hyperhydricity were observed in plantlets where the immersion duration was 120 and 150 s. These findings suggest that an immersion interval of 8 h, combined with an immersion duration of 90 s, could be the optimal setting for growth and development of carnation ‘Dreambyul’ plantlets cultured in the TIS.

     

某些因素对玻璃苗形成的影响和玻璃苗在形态解剖上的特点

瑞香试管苗的玻璃化受到细胞分裂素浓度和茎尖外植体大小的影响,细胞分裂素浓度越高,茎尖外植体越小,产生的玻璃苗越多。玻璃苗与正常试管苗的茎尖在形态结构上有明明显差别,主要表现为顶端分生组织原体原套结构异常和细胞的明显液泡化。     

某些因素对玻璃苗形成的影响和玻璃苗在形态解剖上的特点

瑞香试管苗的玻璃化受到细胞分裂素浓度和茎尖外植体划、的影响,细胞分裂素浓度越高,茎尖外植体越小,产生的玻璃苗越多,玻璃苗与正常试管苗的茎尖在形态结构上有明显差别,主要表现为顶端分生组织原体原套结构异常和细胞的明显液泡化。     

The Use of Single Somatic Embryo Culture in Propagating and Regenerating Lucerne (Medicago sativa L.)

Embryogenic cultures of lucerne (Medicago sativa L.) cv. Robothave been established and propagated on medium containing yeastextract. These cultures consisted of unorganized callus tissuebearing embryogenic centres which increased in size during subculture,yielding new regenerated somatic embryos at the end of each20-d subculture. A development in the propagation of the embryogenic cultureswas the establishment of single embryo culture in hormone-freemedium where, in selected cases, the process of recurrent somaticembryogenesis (RSE) took place on the hypocotyl of explantedembryos. The process was independent of supporting callus tissueand occurred on simple defined medium. Single embryos underwenteither plantlet development or continued RSE on the hypocotyl.One third of the regenerated plantlets showed RSE after thetwo to three trifoliate leaf stage. In these cases shoot developmentstopped and only somatic embryo production took place. In vitrocloning of regenerated plantlets allowed us to reproduce eachparticular genotype before transplantation into soil. Lucerne (alfalfa), Medicago sativa L., somatic embryogenesis, single embryo culture     

Cytokinin Regulation of Flower and Pod Set in Soybeans (Glycine max(L.) Merr.)

Exogenous application of cytokinin to raceme tissues of soybean(Glycine max(L.) Merr.) has been shown to stimulate flower productionand to prevent flower abortion. The effects of these hormoneapplications have been ascertained for treated tissues, butthe effects of cytokinins on total seed yields in treated plantshave not been evaluated. Our objectives were to examine theeffects of systemic cytokinin applications on soybean yieldsusing an experimental line of soybeans, SD-87001, that has beenshown to be highly sensitive to exogenous cytokinin application.Soybeans were grown hydroponically or in pots in the greenhouse,and 6-benzylaminopurine (BA) was introduced into the xylem streamthrough a cotton wick for 2 weeks during anthesis. After theplants had matured, the number of pods, seeds per pod, and thetotal seed weight per plant were measured. In the greenhouse,application of 3.4 x 10^-7 moles of BA resulted in a 79% increasein seed yield compared with controls. Results of field trialsshowed much greater variability within treatments, with consistent,but non-significant increases in seed number and total yieldsof about 3%. Data suggest that cytokinin levels play a significantrole in determining total yield in soybeans, and that increasingcytokinin concentrations in certain environments may resultin increased total seed production. Copyright 2001 Annals ofBotany Company Glycine max, soybean, flower abortion, cytokinin, 6-benzylaminopurine, hydroponic, seed yield, wicking     

Direct Somatic Embryogenesis, Plant Regeneration and Evaluation of Plants Obtained from Mesophyll Protoplasts of Brassica juncea)

Isolated mesophyll protoplasts of Brassica Juncea directly developedinto somatic embryos on medium supplemented with an auxin anda cytokinin. The somatic embryos germinated into plantlets whenmedium was supplemented with GA₂, and ABA. The plantlets weretransferred to the field and the second generation (P₂) wasevaluated for various agronomic characters. Some of the P₂ linesshowed variation in height and time required for flowering.Only two out of 53 lines were comparable to the control in grainyield Brassica Juncea, Indian mustard, direct embryogenesis, protoclonal variation, yield     

A Cytochemical Study of Differentiation and Breakdown of Vessel End Walls

A cytochemical approach was used to study the architecture ofthe end wall and its modifications during vessel differentiationin Populus italica and Dianthus caryophyllus. The combinationof cytochemical techniques with selective extractions to removeend wall subunits provides information on the different componentsof the end wall and on the method of perforation. The end wallappears to be formed mainly from pectins and hemicelluloses.No cellulosic components nor lignin were found though the 3,3'-diaminobenzidine assay revealed the presence of peroxidases.That the method of perforation may depend on the species isdiscussed. Dianthus caryophyllus L., carnation, Populus italica, poplar, xylem vessel, cell wall, cytochemistry, vessel differentiation     

Cryopreservation of Shoot Tips of Tetraploid Potato (Solanum tuberosumL.) Clones by Vitrification

In vitro-grown shoot tips of five tetraploid potato (SolanumtuberosumL.) clones were cryopreserved by vitrification. Excisedshoot tips (0.5–0.7 mm) were pre-cultured on filter paperdiscs over half strength liquid Murashige and Skoog (MS) mediumsupplemented with 8.7 µMGA₃and different combinationsof sucrose (0.3, 0.5 and 0.7M) plus mannitol (0, 0.2 and 0.4M)for 2 d under a 16 h photoperiod at 24 °C. The pre-culturedshoot tips were either successively loaded with 20 and 60% PVS2 solutions or directly exposed to concentrated vitrificationsolution before physical vitrification during liquid nitrogentreatment. The vitrified shoot tips were warmed rapidly andtreated with dilution mixture (MS+1.2Msucrose) for 30 min beforeplating on regrowth medium. Addition of mannitol to the pre-culturemedium improved survival of vitrified shoot tips. Direct dehydrationof pre-cultured shoot tips with concentrated PVS 2 was detrimentalto survival of vitrified shoot tips. Shoot tips pre-culturedon medium containing 0.3Msucrose plus 0.2Mmannitol, and loadedwith 20% PVS 2 for 30 min followed by 15 min incubation in 60%PVS 2 and 5 min incubation in 100% PVS 2 at 0 °C resultedin up to 54% survival after vitrification. About 50% of vitrifiedand warmed shoot tips formed shoots directly. Post-thaw culturingof vitrified shoot tips on medium containing an elevated levelof sucrose (0.2M) under diffuse light for the first week enhancedthe survival rate. Continuous culturing of vitrified shoot tipson high-sucrose medium induced multiple shoot formation.Copyright1998 Annals of Botany Company Solanum tuberosumL., potato, cryopreservation, germplasm conservation,in vitroconservation, meristems, shoot tips, tissue culture, vitrification.     

Micropropagation of Wild Service Tree (Sorbus torminalis [L.] Crantz): The Regulative Role of Different Aromatic Cytokinins During Organogenesis

The influences of three different aromatic cytokinin derivatives [6-benzylaminopurine, meta-topolin, and 6-(3-methoxybenzylamino)purine-9-ß-D-ribofuranoside (MeOBAPR)] on in vitro multiplication and rhizogenesis of the wild service tree (Sorbus torminalis [L.] Crantz) were compared. The highest micropropagation rate (24 new shoots per explant after 3 months of cultivation) was achieved on media containing BAP. On the other hand, the best rooting microcuttings were those multiplied on a medium containing MeoBAPR. To compare these results with the levels of endogenous cytokinins in multiplied explants, a newly developed UPLC-ESI(+)-MS/MS method was used to determine levels of 50 cytokinin metabolites in explants cultivated 12 weeks on media supplemented by BAP and of the two other aromatic cytokinin analogs used. Several significant differences among the levels of endogenous cytokinins, extracted from the explants, were found. The concentration of BAP9G, an important metabolite suspected to be responsible for inhibition of rooting and acclimatization problems of newly formed plantlets, was found to be the highest in microcuttings grown on media supplemented with BAP. This agrees well with the results of our rooting experiments; the lowest percentages of rooted plantlets 6 weeks after transferring shoots on rooting medium were present on explants multiplied on BAP. In contrast, BAP was still the most effective for the induction of bud formation on primary explants. Levels of the most active endogenous isoprenoid cytokinins, tZ, tZR, and iPR, as well as O-glucosides were also suppressed in explants grown on BAP compared with those of explants treated with other cytokinin derivatives. This may be the result of a very high BAP uptake into the explants grown on this cytokinin. On the other hand, endogenous concentrations of cis-zeatin derivatives as well as dihydrozeatin derivatives were not affected. Differences in the production of another plant hormone, ethylene, that plays an important role in controlling organogenesis in tissue culture, were also observed among S. torminalis plantlets grown in vitro on media containing different cytokinins tested. The highest ethylene levels were detected in the vessels containing media supplemented with mT. They were two to four times higher compared with the production by the S. torminalis explants cultivated on other media used. Finally, the levels of free IAA were also determined in the explants. S. torminalis plantlets grown on media containing BAP contained the lowest level of auxin, which is again in good agreement with their loss of rooting capacity. The results found in this study about optimal plant hormone concentrations may be used to improve in vitro rooting efficiency of the wild service tree and possibly also of other plant species.