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.     

Does Lilium bosniacum merit species rank? A classical and molecular-cytogenetic analysis

Four populations of the Balkan endemic taxon Lilium bosniacum from Bosnia and Herzegovina were investigated. Conventional karyological study did not reveal any important differences. Molecular-cytogenetic studies pointed out certain interpopulation and intrapopulation variability, and important differences in organization of ribosomal genes in regard to its closest relative L. carniolicum. The results of fluorochrome banding and FISH experiment for L. bosniacum were reported here for the first time. Differences occurred in the number and position of 18S-5.8S-26S ribosomal gene loci for some individuals from population Kladanj. Heterochromatic bands revealed with DAPI after FISH experiment were constant. All investigated populations possess the same number of active NORs except some individuals from Kladanj population. Genome size and GC-bases percentage, estimated by flow cytometry, did not show any significant difference among the populations. However, the present results reveal clear interspecific differentiation between two endemics, L. carniolicum and L. bosniacum.     

Secondary metabolite content in rhizomes, callus cultures and in vitro regenerated plantlets of Solidago chilensis

An in vitro culture system leading to the formation of callus and plant regeneration, starting from nodal sections and shoot tips, was developed for Solidago chilensis (Asteraceae). The content of the gastroprotective diterpene solidagenone as well as the phenolics chlorogenic acid (CA) and rutin was determined either in rhizomes from wild growing plants and in callus and in in vitro regenerated plantlets by analytical HPLC. Additionally, total phenolic and flavonoid content was assessed in plant samples, callus and cell suspensions. In terms of dry starting material, the percentual solidagenone content in nine S. chilensis samples ranged from 0.5-3.5% for rhizomes from wild growing plants, 0.1-0.3% for callus and 0.3% for an in vitro regenerated plantlet, respectively. The highest solidagenone contents were found in the wild plant during the late summer in the months of March and April (3.5-2.2%) while highest values for chlorogenic acid (0.5%) and rutin (0.4%) were detected in May, before senescence. The callus tissue and cell suspensions contained some 1.8-2.0 and 1.2% of total phenolics, respectively. CA was the main phenolic in the cell suspension while only traces were found in the callus. Rutin was not detected in the callus nor cell culture.     

In vitro regeneration of loblolly pine and random amplified polymorphic DNA analyses of regenerated plantlets

 Adventitious buds were induced from organogenic callus derived from mature zygotic embryos of three lines (E-311, E-440, and E-822) of loblolly pine (Pinus taeda L.) within 27 weeks of culture. The influence of cytokinins, silver nitrate, and low-temperature treatment on the differentiation of adventitious buds was analyzed. Elongation of adventitious buds was achieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid (IBA) and 1 mg/l 6-benzyladenine (BA). After adventitious shoots had rooted on TE medium supplemented with 0.5 mg/l IBA, 2 mg/l BA, and 0.5 mg/l gibberellic acid, 498 regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1 :>: 1) soil mixture; 351 of these survived in the field. Total DNA was extracted from 21 regenerated plantlets randomly chosen from the 151 regenerated plantlets of line E-822. Random amplified polymorphic DNA (RAPD) analysis using 80 arbitrary oligonucleotide 10-mers showed that 21 primers gave 107 clear reproducible bands, with the amplification products being monomorphic for all of the plantlets of line E-822 tested. A total of 2,247 bands obtained from these studies exhibited no aberration in RAPD banding patterns among the tested plantlets. These results suggest that somatic organogenesis can be used for clonal micropropagation of some lines of loblolly pine without the fear of the appearance of unwanted somaclonal variants. Received: 5 August 2000 / Revision received: 5 September 2000 / Accepted: 10 October 2000     

Ploidy stability in embryogenic cultures and regenerated plantlets of tamarillo

Ploidy levels of short-term (1 and 2 years) and long-term (7 and 10 years) embryogenic cultures as well as of regenerated plantlets of tamarillo were analyzed by flow cytometry and chromosome counts. Embryogenic cultures were induced from expanding leaves cultured in the presence of Picloram or 2,4-dichlorophenoxyacetic acid (2,4-D) and monthly subcultured on the same media. Embryo development and plantlets were obtained following subculture of the embryogenic tissue in auxin free medium containing gibberellic acid (GA₃). Seedlings and rooted shoots from axillary shoot proliferation were used as controls. The results showed that in long-term embryogenic cultures the ability to develop somatic embryos and plantlets was reduced. Embryogenic tissues maintained for 10 years were mostly aneuploids of the tetraploid (2n = 4x = 48) level whereas those kept in culture for 7 years or less were also mostly aneuploids but of the diploid (2n = 2x = 24) level. The results obtained by flow cytometry were, in general, consistent with those obtained by chromosome counts. The chromosome alteration observed in the embryogenic tissues was already present after 1 year of culture and increased with culture age, hence impairing the maintenance of these tissues for long periods without affecting chromosome stability of the regenerated plantlets. However, the occurrence of triploids and tetraploids as well as aneuploids can be useful for breeding purposes. A value around 23 pg/2C was found for the genome size of tamarillo largely exceeding the value previously published (15.50 pg/2C).     

High-frequency plant regeneration via somatic embryogenesis and organogenesis and in vitro flowering of regenerated plantlets in Panax ginseng

 The morphogenesis ability of light yellowish globular callus derived from cotyledons of mature zygotic embryos of Panax ginseng was investigated. The optimal media for somatic embryogenesis and shoot organogenesis were MS medium containing 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid, 0.1 mg l^–1 6-benzyladenine (BA), and 500 mg l^–1 lactoalbumin hydrolysate, and SH medium supplemented with 0.5 mg l^–1 α-naphthaleneacetic acid, 0.1 mg l^–1 BA, and 500 mg l^–1casein hydrolysate. The influences of glucose, mannose, fructose, and sorbose in the media on somatic embryogenesis and shoot organogenesis were revealed as differences in the numbers of somatic embryos and adventitious shoots per gram of morphogenic callus. The best regeneration of somatic embryos was obtained on medium containing glucose, with a mean of 8.7 somatic embryos per gram of callus. The best regeneration of shoots was observed on medium containing fructose, with an average of 12.2 adventitious shoots per gram of callus. Of the somatic embryos 95% were converted into regenerated plantlets, and 100% of adventitious shoots rooted to form regenerated plantlets. Regenerated plants were successfully established in soil. Flowering was observed in 5.7% of the regenerated plants derived from shoot organogenesis and in 1.4% of the regenerated plants derived from somatic embryogenesis. Received: 1 December 1998 / Revision received: 13 September 1999 / Accepted: 20 September 1999     

Sugarcane mosaic virus in plantlets regenerated from diseased leaf tissue

Plantlets produced from sugarcane leaf tissue were examined to determine the effect of propagation on the frequency of occurrence of sugarcane mosaic virus (SCMV).Explants from immature leaf tissues of the sugarcane variety CP 72-356 (Saccharum interspecific hybrid), healthy or SCMV-infected, were cultured on Murashige-Skoog medium to which a combination of cytokinin and auxin had been added. Plantlets developed on healthy and infected leaf tissue within 6 weeks. The juice from plantlets was assayed for SCMV on Rio sorghum (Sorghum bicolor (L.) Moench, var. Rio) seedlings and on sugarcane varieties CP 31-294 and CO 31-588 for SCMV-strain identification. Results indicated that SCMV strain H was transmitted from the donor tissue to the regenerated plantlets. Observation on plantlets reared in the greenhouse showed that 23% had symptoms of SCMV. In a second replicated experiment, the leaf tissue from plants of POJ 234 free of mosaic or infected with SCMV strain A, B, D, H, or I was cultured. Each of the five strains was transmitted from donor to plantlet as indicated by assays on sorghum and sugarcane varieties. From 11 to 88% of the plantlets had mosaic symptoms, depending on the strain infecting the donor plant. In this experiment, SCMV-strain M was transmitted from an unidentified donor variety to 23% of the regenerated plantlets.Portions of this paper have been presented to the American Society of Sugar Cane Technologists, at the meeting in Clearwater, Florida in June, 1984.     

Comparison of somatic embryogenesis-derived coffee (Coffea arabica L.) plantlets regenerated in vitro or ex vitro: morphological,mineral and water characteristics

Coffea arabica L. plantlets obtained ex vitro after sowing somatic embryos produced in a bioreactor in horticultural substrate were compared with those obtained in vitro from the same embryo population under conventional culturing conditions on semi-solid media. The intensity and quality of aerial and root system development were compared. Shoot emergence was more efficient in vitro but rooting frequencies were low. In contrast, all ex vitro-regenerated embryos rooted. The cotyledon area of mature embryos produced in a bioreactor positively affected plantlet development when regeneration was carried out ex vitro. Embryos with an intermediate cotyledon area (0.86 cm2) had the highest rates of plant conversion ex vitro (63%), and also resulted in vigorous plantlets. Mortality was higher in nursery conditions, but better plant development was obtained. The quality of plantlets produced under ex vitro conditions was reflected in better growth of the aerial and root systems, and also by similar morphological, mineral and water status characteristics to seedlings. Unlike roots formed on semi-solid media, those produced in soil were branched, fine (30-50% had a diameter of less than 0-5 mm) and they bore root hairs. Leaves of plantlets regenerated ex vitro had a histological structure similar to that of seedling leaves, and a lower stomatal density (100 vs. 233 mm-2). Moreover, they were more turgid, as indicated by higher pressure potential (psiP) (0.91 s. 0.30 MPa) and relative water content values (97 vs. 93%). Furthermore, under in vitro conditions, leaves had larger stomata which were abnormally round and raised. Direct sowing of germinated somatic embryos resulted in the rapid production of vigorous plantlets under ex vitro conditions, whilst removing the need for problematical and costly conventional acclimatization procedures.     

Micropropagation ofPanax notoginseng by somatic embryogenesis and RAPD analysis of regenerated plantlets

Somatic embryogenesis was induced in callus tissues derived from young flower buds ofPanax notoginseng via callus within 18 weeks of culture. The mature somatic embryos were germinated on half-strength Murashige and Skoog's (MS) medium supplemented with gibberellic acid A₃(GA) and 6-benzyladenine (BA). The most suitable medium for optimal root formation proved to be MS medium supplemented with 1-naphthaleneacetic acid (NAA). Total DNA was extracted from the leaves of the regenerated plantlets ofP. notoginseng. Analysis of random-amplified polymorphic DNA (RAPD) using 21 arbitrary oligonucleotide 10-mers, showed the genetic homogeneity ofP. notoginseng. The amplification products were monomorphic for all of the plantlets ofP. notoginseng regenerated by embryogenesis, suggesting that somatic embryogenesis can be used for clonal micropropagation of this plant.     

Expression of hormone-autotrophy in tissues of plantlets regenerated from habituated callus of Nicotiana bigelovii

Experiments presented in the literature show that habituation, a phenomenon characterized by a newly acquired capacity of in vitro cultured plant cells to produce growth regulatory substances, occurs at high rates, leaves the heritably altered cells totipotent and is regularly reversible. Many evidences show that habituation has an epigenetic basis. We report here that habituation in Nicotiana bigelovii var. quadrivalvis tissue cultures is a persistent character not reversed by plant regeneration under the conditions tested.     

二色补血草试管苗生根及移栽基质研究

以二色补血草为材料,研究了不同浓度激素配比对试管苗生根量及根长的影响,以及生根苗在不同移栽基质中的成活率和生长状况。结果表明,试管苗在MS+NAA 1.0mg/L培养基中的平均根量最多,在MS+IBA 1.0mg/L培养基中平均根长最长;生根苗在泥炭+珍珠岩(1:1)的基质中生长最好。同时进行降低成本的试验。     

Production of eleutherosides in in vitro regenerated embryos and plantlets of <Emphasis Type="BoldItalic">Eleutherococcus chiisanensis</Emphasis>

High frequency somatic embryogenesis of Eleutheorcoccus chiisanensis was achieved through suspension culture of embryogenic cells in hormone-free Murashige and Skoog liquid medium supplemented with 30 g sucrose l⁻¹. Cotyledonary somatic embryos were germinated and converted into plantlets using 20 μM gibberellic acid which were then grown in a 10 l airlift bioreactor. HPLC analysis revealed the accumulation of eleutheroside B, E and E1 in the embryos and plantlets. Thus mass production of embryos and plantlets of E. chiisanensis can be achieved in liquid cultures and the biomass produced may become an alternative source of eleutherosides.     

Direct and indirect organogenesis of Clivia miniata and assessment of DNA methylation changes in various regenerated plantlets

Clivia miniata is an important indoor ornamental plant and has been reported to have medicinal value. We developed an efficient in vitro micropropagation protocol from young leaves (indirect organogenesis), young petals (indirect organogenesis) and shoot tips (direct organogenesis) of this plant. Using young leaves and shoot tips as explants, the regeneration frequencies were much higher than those in previous investigation and the regeneration was dependent upon less nutrition. We speculated that the leaf-derived callus can generate amino acids necessary for protein synthesis by itself. We employed the methylation-sensitive amplified polymorphism (MSAP) method to assess cytosine methylation variation in various regenerated plantlets and between organs. The MSAP profiles indicated that the frequency of somaclonal variation in the form of cytosine methylation was highest in petal-derived plantlets followed by secondary leaf-derived, primary leaf-derived and shoot tip-derived plantlets, but the methylation variation in petal-derived plantlets was lower than between petals and leaves of a single plant. The results indicated that the methylation variation in regenerated plantlets was related to the types of explants, regeneration pathways and number of regeneration generations. Two possible factors for the highest somaclonal variation rate in petal-derived plantlets are the callus phase and petal-specific set of epigenetic regulators. The property of meristem integrity can account for the lowest variation rate in shoot tip-derived plantlets. Moreover, the secondary plantlets underwent a longer total period of in vitro culture, which can explain why the methylation variation rate in the secondary plantlets is higher than in the primary ones. KEY MESSAGE: Methylation variation in regenerated plantlets of C. miniata was found to be related to the types of explants, regeneration pathways and number of regeneration generations.     

Micropropagation of Bambusa edulis through nodal explants of field-grown culms and flowering of regenerated plantlets

Nodal explants obtained from 10-year-old field-grown culms of Bambusa edulis produced multiple shoots on a Murashige-and-Skoog-based medium supplemented with 0.1 mg/l of thidiazuron (TDZ). Hundreds of regenerated shoots rooted well on a medium supplemented with 0.01 mg/l TDZ and 0.5 mg/l 2,4-dichlorophenoxyacetic acid and were successfully transferred to soil for field trials. Albinism occurred at the rate of about 30% among the regenerated shoots, and isolated albino shoots also proliferated on the medium containing TDZ. Some of the green and albino shoots also flowered on the medium containing TDZ. A potted plant also flowered and survived after flowering. Received: 20 August 1997 / Revision received: 12 December 1997 / Accepted: 12 January 1998     

In vitro regeneration of plantlets in Brassica alboglabra

Different vegetative parts of Brassica alboglabra seedlings and mature plants were used as explants in culture.A high frequency (60–100%) of shoot regeneration was obtained from hypocotyl explants, nodal stem segments, internodal segments and shoot apices cultured on Murashige-Skoog basal medium. Addition of 6-benzylaminopurine and kinetin increased the average number of shoots per explant. When detached and transferred to basal medium, the shoots readily developed roots. Regenerated plantlets could be successfully transplanted in soil.     

Nuclear cytology of callus and plantlets regenerated from pea (Pisum sativum L.) meristems

Summary The chromosomal status of calli and plantlets regenerated fromPisum sativum shoot apical meristems was studied. Chromosome mosaicism (aneusomaty) occurs during callus induction and proliferation, mostly owing to nuclear fragmentation prior to mitosis in the first days of culture. Plantlets regenerated from calli are diploid or aneusomatic, but a selective advantage of diploid cells (diplontic selection) takes place with plantlet growth. The results are discussed in relation to the possibility of inducing chromosomal and/or genetic variability by using meristematic tissues as expiants.     

Genetic analyses of micropropagated and regenerated plantlets of banana as assessed by RAPD and ISSR markers

A cultivar of dessert banana, namely, Nanjanagudu Rasabale (NR), classified under group “silk” (of genotype AAB), is seriously under the threat of extinction due to its susceptibility to bacterial wilt and bunchy-top virus disease. A regeneration protocol using tissue culture method was developed (Venkatachalam et al. 2006), where a large number of plantlets were regenerated from leaf base explants. Simultaneously, a micropropagation protocol was also developed where high levels of up to 53.28 μM of benzylamino purine (BAP) and 55.80 μM of kinetin (Kn) were used. The progressive increase of cytokinins levels resulted in concomitant increase in shoot number, with a maximum of 80 shoot buds per segment in BAP (31.08 μM). The plantlets were analyzed for their genetic stability using randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. A total of 50 RAPD and 12 ISSR primers resulted in 625 distinct and reproducible bands showing homogeneous RAPD and ISSR patterns. Band intensity histogram of each gel confirmed their monomorphic nature with no genetic variation among the plantlets analyzed. The present study has established for the first time that the regeneration and rapid micropropagation protocol developed through the present study will be of great use in conserving the endangered cultivar – NR – without risk of genetic instability.     

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.     

In vitro regeneration of Hypoxis colchicifolia plantlets

Corms of Hypoxis species are heavily traded for use in traditional medicine in southern Africa. High demand has increased unsustainable harvesting from the wild, diminishing natural populations. Micropropagation of Hypoxis colchicifolia was investigated as a means of mass producing plants for both commercialization and re-establishment in the wild. Various plant organs were tested as explant sources and decontamination optimized for each explant type. Seeds failed to germinate in vitro, and inflorescence peduncles, leaves (young and mature) turned brown and did not respond in culture. Only 6% of corm explants produced callus and shoots. Flower buds responded best, with multiple shoots initiated from explants either directly from meristemoids or indirectly via callus. The problem of browning due to phenolic exudation was solved by including polyvinylpyrrolidone (PVP) in the culture medium when required. Common pathogens were partially controlled by washing affected explants in benomyl solutions. Rooting and corm induction were successful, and plantlets could be stored at low temperature (10 °C) prior to acclimatization with no adverse effects. In planting trials with 5-month-old and 21-month-old plants regenerated using the improved protocols, flowering percentage, corm and leaf size were increased significantly in plants grown in pots compared to those grown in the field over a 28 month period.