ABA content and sensitivity during the development of dormancy in lily bulblets regenerated in vitro

We measured ABA content and sensitivity in bulblels of Lilium speciosum Thunb , regenerating from scale explants in vitro at temperatures (15, 20 or 25°C) that allowed the development of various levels of dormancy (very low, intermediate or high, respectively). The one-step purification and the accuracy of the immunoassay were confirmed by HPLC and by liquid chromatography/mass spectrometry. ABA content was not correlated with dormancy development. Sensitivity to ABA was determined as the difference in sprouting performance of excised bulblets on medium with and without ABA. In bulblets regenerating at 20 or 25°C. ABA sensitivity was high during the period of dormancy establishment and decreased thereafter. Dormant hulblets were almost completely insensitive to ABA. The changes in sensitivity to ABA were confirmed by measuring the level of ABA in bulblets at the time of sprouting. This level was, as expected, highest in bulhlels with low ABA-sensitivity. Briefly cold-treated bulblets, in which dormancy may he re-established by culture at 20°C, again became sensitive to ABA. ABA sensitivity decreased with increasing temperature bulblets that regenerated at I5°C and hardly developed any dormancy, were very sensitive to ABA. It was concluded that in addition to ABA sensitivity another, still unknown, factor played a key role in dormancy development.     

Effect of low temperature on dormancy breaking and growth after planting in lily bulblets regenerated in vitro

Lilies regenerating on scale segments may develop dormancy in vitro depending on the culture conditions. The dormancy is broken by storage for several weeks at a low temperature (5 °C). The effect of the low temperature on sprouting, time of leaf emergence and further bulb growth was studied. Dormant and non-dormant bulblets were regenerated in vitro on bulb scale segments cultured at 20 °C or 15 °C, respectively. The low temperature not only affected the number of sprouted bulblets but also the time of emergence. The longer the cold storage, the faster and more uniform leaf emergence occurred. Both dormant and non-dormant bulblets grew faster after a low temperature treatment of six weeks. Thus, during dormancy breaking the tissue is prepared not only for sprouting but also for subsequent bulb growth. These processes are rather independent as low temperature stimulates growth in non-dormant bulblets whereas these bulblets sprout also without treatment at low temperature. Moreover, the hormone gibberellin induces rapid sprouting but has no influence on further bulb growth. Good growth in bulblets exposed to the low temperature coincided with production of an increased leaf weight. However, the relationship is not absolute as bulblets that were cold-treated for six weeks grew larger than bulblets cold-treated for four weeks but the formation of leaf biomass was similar. During storage at low temperature starch was hydrolyzed in the bulb scales and sugars accumulated. This indicates that during this period, preparation for later bulb growth involves mobilization of carbohydrate reserves which play a role in leaf growth and development of the photosynthetic apparatus. Starch hydrolysis proceeded in the outer scales after planting. Approximately six weeks later, the switch from source to sink took place in the bulblet, which became visible as a deposition of starch in the middle scales.     

The role of scale explants in the growth of regenerating lily bulblets in vitro

Plant Cell, Tissue and Organ Culture (PCTOC) - Lily scale-explants cultured in vitro regenerate adventitious bulblets at their base. Large scale-explants (6?×?18 mm; the...     

Development of dormancy in different lily genotypes regenerated in vitro

Dormancy development in four Lilium genotypes,L. speciosum, Star Gazer, C. King and Snow Queenregenerated in vitro was compared. Major factorsinfluencing dormancy development were the same for different genotypes andespecially L. speciosum and Star Gazer, that are closelyrelated, reacted similarly. Temperature was the main factor in dormancyinduction and breaking. The range of temperatures that induced dormancy and thelevel of dormancy that developed differed per genotype. In Star Gazer, dormancydeveloped gradually but in Snow Queen, dormancy developed very fast. Thereactions to temperature, reflected the climate in the area of origin. Abscisicacid deepened the level of dormancy induced by temperature but had no effectunder non-inductive temperature conditions. When abscisic acid synthesis wasblocked, no dormancy developed. Dormancy in all genotypes was broken by coldincubation for severalweeks. The cold requirement of the genotypes differed in line with the naturalwinter conditions in their habitat. The effect of hormones on dormancy breakingwas also investigated. A gibberellin treatment of 24 h brokedormancy in L. speciosum, Star Gazer and Snow Queen.     

Abscisic acid controls dormancy development and bulb formation in lily plantlets regenerated in vitro

Plantlets of lily regenerated in vitro from scale explants consist of scales and leaves from which the base of the petiole has swollen to a scale. Fluridone, an inhibitor of ABA-synthesis, applied during culture in vitro, inhibited the swelling of the petioles and promoted leaf formation. At high fluridone concentrations (10 or 33μ M ), swelling was completely blocked, and plantlets consisted of leaves only. Addition of ABA during the regeneration in vitro had the opposite effect and resulted in plantlets with scales only. When applied simultaneously with fluridone, ABA nullified the effect of fluridone. This demonstrates that bulb formation in lily is under the control of ABA. Lily plantlets regenerated in vitro on scale explants at 20 or 25°C were harvested after 11 weeks, and the leaves were removed from the bulblets. The bulblets were dormant and required a cold treatment to achieve rapid emergence after planting in soil. Fluridone added during the culture in vitro prevented the development of dormancy, and the bulblets did not require a cold treatment. The effect of fluridone was nullified by simultaneous addition of ABA. Bulblets harvested after 6 weeks of culture at 20°C had not yet developed dormancy. Bulblets regenerated at 15°C were only slightly dormant. In both types of bulblets, it is unlikely that the lack of dormancy was due to low ABA-levels since addition of ABA did not affect the dormancy status. These data indicate that the level of endogenous ABA and an unknown additional factor play major roles in the development of dormancy.     

Regeneration of bulblets from twin scales ofCrinum macowanii in vitro

Tissue culture techniques were applied to study the regeneration and growth of bulblets from bulb scale segments ofCrinum macowanii Bak. (bush- or march lily)in vitro. Shoots were induced on twin scales taken from the basal plate region of flowering-size bulbs on Murashige and Skoog (MS)-medium containing 0–20 mg l^–1 NAA and BA and a modified MS medium (MMS medium) containing 1.25 mg l^–1 ancymidol (A-Rest^TM), 0.1 mg l^–1 NAA and 0.1 mg l^–1 kinetin (ANK). Large bulblets could only be initiated on the latter. Subsequently the bulblets of 5 mm or more in diameter were trimmed and split in half, and secondary plantlets were regenerated on MMS-medium containing ANK or MS-medium without any growth regulators which in turn grew into bulblets suitable for splitting within 12–16 weeks. A total of 700–1000 bulblets could be obtained from each initial bulb within 12 months. Anatomical studies showed that the shoots were initiated from the epidermis and hypodermis on the abaxial surface of the meristematic tissue of the basal plate of the bulb scale. This technique is useful for the multiplication and preservation of a genotype, since plantlets regenerated in this manner should be genetically uniform.     

Contribution of explant carbohydrate reserves and sucrose in the medium to bulb growth of lily regenerated on scale segments in vitro

Bulb size is an important factor determining phase change in Lilium : phase change only occurs in bulblets over a certain threshold weight. After phase change has occurred, bulblets sprout with a stem with many leaves. Juvenile bulblets sprout with only a few leaves. The factors contributing to bulb size were studied during in vitro regeneration of bulblets on scale segments. The larger the explants, the larger the regenerated bulblets. Explant size influenced bulb growth during the complete culture period. Bulb growth was stimulated by a high sucrose concentration. The contribution of the medium and the explant reserves to bulb growth were studied in large and small explants using labelled sucrose. Sucrose was mainly taken up through the cut surfaces. In freshly cut explants, the rate of uptake was correlated with the size of the contact area, but at later stages, when regenerating organs were present, the difference in uptake rate of small and large explants almost disappeared. Small explants had a larger sink activity than large ones. Explants with regenerating organs took up more sucrose than freshly cut explants. Sucrose uptake and bulb growth were rather constant in the later phases and doubled when the sucrose concentration was doubled. Partitioning of label from the sucrose over the various organs, was also rather constant in time: approximately 25% was accumulated in the bulblets, 35–45% in the explant and 30–35% was converted to CO₂. About half of the label in the explant was recovered at the proximal side where regeneration takes place. Sucrose, once incorporated in the storage pools in the explant, either remained in the explant or was converted to CO₂. Redistribution to the growing bulblets hardly occurred. The percentage of bulb growth that could be attributed to uptake of medium components was constant over the regeneration period: 45–50% for large and 65–75% for small explants.     

Formation of onion bulblets in vitro and viability during medium-term storage

 The influences of light conditions, sucrose and ethylene on in vitro formation and storability of onion (Allium cepa L.) bulblets were studied in various accessions. Light, sucrose and ethylene influenced bulb formation. Storability was primarily enhanced by a high sucrose concentration (100 g/l) in the culture medium. The bulbing process was characterised by changes in bulbing ratio, leaf length, number of leaves and leaf development time. The viability of bulbs after 1 year of in vitro storage at low temperatures was determined by their growth reaction in subsequent subcultures, growth after transfer into the greenhouse and tetrazolium staining. Sufficient sprouting of bulblets previously stored at –1  °C demonstrated the possibility of storing them in a low-temperature, slow-growth culture. Received: 8 June 2000 / Revision received: 5 October 2000 / Accepted: 5 October 2000     

The development of dormancy in bulblets of Lilium speciosum generated in vitro

We have studied the effect of various in-vitro conditions on dormancy of bulblets generated on scale explants of Lilium speciosum Thunb. cv. Rubrum nr. 10. The bulblets were harvested after 11 weeks of culture. Dormancy was measured by determining the percent emergence in soil of viable, non-cold-treated bulblets. A study of the physical conditions showed that temperature had a strong effect on the induction of dormancy (15°C induced hardly any dormancy; 25°C induced a high level of dormancy), whereas short or long day and light or dark had no effect. Of the medium components, a low concentration of sucrose (1 gl^–1 or less) or a high concentration of gibberellic acid (1 mg 1^–1) reduced the level of dormancy. Application of various concentrations of abscisic acid, 6-benzylaminopurine, -naphthaleneacetic acid, indole-3-acetic acid, 2,3,5-triiodobenzoic acid or a Murashige and Skoog macro- and microelement mixture did not affect the dormancy status.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige & Skoog macro- and microelements - NAA -naphthalene-acetic acid - TIBA 2,3,5-triiodobenzoic acid     

Frictional properties of regenerated cartilage in vitro

Although tribological function is the most important mechanical property of articular cartilage, few studies have examined this function in tissue-engineered cartilage. We investigated changes in the frictional properties of cartilage regenerated from the inoculation of rabbit chondrocytes into fibroin sponge. A reciprocating friction-testing apparatus was used to measure the friction coefficient of the regenerated cartilage under a small load. The specimen was slid against a stainless steel plate in a water vessel filled with physiological saline. The applied load was 0.03 N, the stroke length was 20 mm, and the mean sliding velocity was 0.8 mm/s. The friction coefficient of the regenerated cartilage decreased with increasing cultivation time, because a hydrophilic layer of synthesized extracellular matrix was formed on the fibroin sponge surface. The friction coefficient of the regenerated cartilage was as low as that of natural cartilage in the early stages of the sliding tests, but it increased with increasing duration of sliding owing to exudation of interstitial water from the surface layer.     

Factors influencing T-DNA transferring into pollen of lily in vitro

Direct pollen transformation method improves the classical transformation procedures because some tissue culture steps and subsequent regeneration can be avoided. A critical step in the development of Agrobacterium-mediated transformation is the establishment of optimum conditions for T-DNA delivery into tissue. The pollen grains of David lily (Lilium davidii Duchartre) are transformable by Agrobacterium during their germination, and extremely high GUS expression frequency of pollen had been achieved (92.7 ± 2.7%), but not for the ungerminated pollen. The culture medium, Agrobacterium cell density, duration of co-cultivation, and the combination of bacterial strains and plasmids should be optimized to get the highest transformation frequency. Thus, a method for pollen monocotyledonous species reproductive tissues transformation by Agrobacterium in monocots has been successfully developed. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 3, pp. 475–480 The text was submitted by the authors in English.     

Histological characterization of in vitro regenerated structures of Panax ginseng

Callus was induced from mature leaf lamina, petiole, stem, and main and branch root of Panax ginseng and maintained under different nutrient and light conditions. Heterogenous callus cultures differentiated organoids and/or embryoids. Embryoids that arose from callus cultures of leaf origin germinated into shoots. The occurrence of the early stages of different regenerated structures was proved on histological level.     

Characteristics of teratomas regenerated in vitro from octopine-type crown gall

Crown galls induced by infection of tobacco plants with Agrobacterium tumefaciens strain C58-Cl(pTiB6S3) were excised and cultured in vitro. After about one year of culture on medium-lacking phytohormones, two noncloned lines spontaneously formed shoots. Leaf explants from shoots of tumor-line T5 were capable of growing on hormone-free medium, and the resulting mixture of organized and unorganized tissue synthesized octopine. Detached leaves from T5 shoots also synthesized octopine. These results establish that shoots from this octopine-type tumor contain transformed cells and are true crown-gall teratomas.     

Loss of resistance to Colletotrichum trifolii in in vitro regenerated alfalfa

Alfalfa plants were regenerated from callus cultures of three source plants that differed in resistance to anthracnose, caused by Colletotrichum trifolii. All regenerant plants were evaluated for variation in resistance to disease caused by races 1 and 2 of the pathogen. Of eighty-two plants that were regenerated and evaluated, no plants responded differently to inoculation with race 1 of C. trifolii, but two plants (2.4%) differed in resistance when inoculated with race 2. The source plant of these regenerants was resistant to races 1 and 2 of the pathogen but the regenerants were resistant to race 1 and susceptible to race 2. No variants to race 1 were detected. The susceptible response of the variant plants to race 2 was confirmed by cytological analysis and was consistent with the response of nonregenerant susceptible plants. These plants represent a near-isogenic plant model for studying the molecular biology of resistance and susceptibility to anthracnose of alfalfa.     

Phase change enzyme immunoassay

A novel enzyme-linked immunoassay employing a partitioning chromophore was developed. The assay system consisted of an aqueous phase and an immiscible organic solvent. Antigen-antibody interaction was indicated by transfer of a chromogenic indicator from the aqueous phase to an organic layer. The indicator employed was a water-soluble phosphate ester of phenylazophenol. Hydrolysis of the ester by acid or alkaline phosphatase produced a water-insoluble phenol that partitioned into toluene. The enzyme employed in this assay format can be covalently linked to antibody or a specific antibody for the phosphatase can be used. Phase change immunoassays were developed for the measurement of alkaline phosphatase, human IgG in whole blood, and the human tumor marker prostatic acid phosphatase. Solid supports of small polystyrene latex particles and Sephadex were employed.     

Chloroplast ultrastructure of Hypericum perforatum plants regenerated in vitro after cryopreservation

The ultrastructure of leaf mesophyll cells of in vitro cultured Hypericum perforatum L. plants regenerated after cryopreservation was studied. Electron microscopy analysis revealed that the chloroplasts in plants pretreated with abscisic acid and regenerated after cryopreservation were round, with increased amount of starch, rather small volume of the thylakoid system, and destroyed envelope. Plants pretreated with 0.3 M mannitol and cooled at rates of 0.1 or 0.3 °C min^?1 possessed chloroplasts with high starch content that resulted in a reduction of a membrane system. However, the pretreatment with 0.3 M mannitol and cooling at a rate of 0.2 °C min^?1 was the best as chloroplast ultrastructure resembled the controls regenerated without cryopreservation.     

The development of dormancy in bulblets of Lilium speciosum generated in vitro. II. The effect of temperature

Upon harvest, lily ( Lilium speciosum Thunb. cv. Rubrum) bulblets generated in vitro under standard conditions (11 weeks at 20°C) were dormant and needed a cold treatment prior to planting. During culture in vitro at 20°C, the bulblets proceeded through three phases: (1) at first they were non–viable and non-dormant (up to 5 weeks), (2) then viable and non-dormant (5–9 weeks) and (3) finally viable and dormant (from 9 weeks onwards). At 15°C, the bulblets became viable but did not develop dormancy, even after protracted culture. The results suggest that the development of dormancy depends upon an accumulation of'heat units'occurring at temperatures higher than 15°0. At 25°C, the succession of the three phases occurred more rapidly than at 20°C and heat units were accumulated more rapidly. During the third period, the chilling requirement increased showing that heat units continued to be accumulated during this period.
Dormancy connotes an arrest of growth. In lily bulblets, however, the number of scales continued to increase after the induction of dormancy at 20 or 25°C. Many of the scales initiated before the onset of dormancy were formed by swelling of a petiole, whereas, after the onset of dormancy, all scales were formed directly from a primordium. We conclude that the development of dormancy corresponds to a switch in the development of the primordium. Thus, after the induction of dormancy the primordium lost the ability to become a leaf and always developed into a scale.     

Biochemical status of in vitro regenerated Lilium bosniacum and Lilium cattaniae plantlets

Lilium cattaniae (Vis.) Vis. and Lilium bosniacum (G. Beck) Beck ex Fritsch, endemic species of Balkan Dinaric Alps, were micropropagated from seeds collected from their natural habitats. The relationship between peroxidase activity, photosynthetic status and differentiation of Lilium cattaniae and L. bosniacum in vitro was investigated. Peroxidase activity recorded for somatic embryos of Lilium cattaniae obtained on Murashige and Skoog (MS) medium containing 9.05 mM 2.4-dichlorophenoxyacetic acid (2.4-D) and 4.44 mM N6-bezyladenin (BA), was about two times higher than for any other treatment. Photosynthetic status of plantlets obtained through regeneration was explant-specific and generally higher for plantlets regenerated from basal leaf explants than from bulb explants. The accumulation of anthocyanin was detected in some regenerated shoots and more often in plantlets obtained through regeneration from bulb explants. High frequency of somatic embryo formation was recorded for L. cattaniae on MS medium containing 9.05 mM 2.4-D and 4.44 mM BA. The peroxidase activity for L. bosniacum plantlets obtained through regeneration showed treatment-specific elevations. We consider that basal leaf parts are suitable for successful regeneration of these two lilies and that elevation in peroxidase activity is a good indicator of somatic embryogenesis in both lilies.     

Some factors affecting root formation onin vitro regenerated pea shoots

The rooting ability of 2 cm long shoots ofPisum sativum L., derived from differentin vitro shoot-tip cultures in two pea cultivars Bohatýr and Kleine Rheinländerin was evaluated. In three mutually independent experiments the full and half-strength Murashige-Skoog medium (containing full or half concentration of macro and microelements), with sucrose concentrations 10–30 g l-¹, and with various NAA and IAA combinations, was tested. The variant with half concentration of macro- and microelements, supplemented with 30 g l¹ sucrose, and with growth regulators in the quantity of 1 μM proved optimum.     

Application of luminescence for quality evaluation of in vitro regenerated strawberry plants

The parameters of chlorophyll a fluorescence induction were measured: F_v/F_m, S_c/F_m, Rf_d and coefficient of L_d delayed luminescence decay kinetics, related with a course of primary photosynthesis reactions on leaves of strawberry plants, cultured in vitro by means of the micropropagation methods. Strawberry plants cv. Ananasowa from in vitro cultures in optimal condition show significantly higher values of luminescence parameters indicating better condition of plants of this variety in comparison with the variety Senga Sengana. After temperature lowering, however, these values were more reduced than for plants of Senga Sengana, which can be interpreted as higher susceptibility of this variety to chill. Addition of BAP caused disturbance of primary photosynthesis reactions rate, particularly in lower temperature. Auxin 2,4-D had no effect on the luminescence parameters in comparison with control cultures. Dehydration stress strongly diminished the values of measured parameters for Ananasowa variety what indicates the inhibition of primary photosynthesis reaction in leaves. The old culture of Senga Sengana variety showed higher tolerance on linuron in comparison with the new one.