Factors affecting plantlet regeneration from <Emphasis Type="Italic">in vitro</Emphasis> cultured immature embryos and cotyledons of <Emphasis Type="Italic">Prunus mume</Emphasis> “Xue mei”

Using immature embryos and cotyledons as explants, a successful system to culture immature embryos and induce direct regeneration from cotyledons was established for Prunus mume “Xuemei”. For immature embryo culture, a high frequency of plantlet formation (89.5%) from the embryonic axis was obtained using half-strength Murashige and Skoog (1/2 MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). Shoots formed directly from cotyledons with the embryo axis intact when explants were cultured on 1/2 MS medium containing 2.2 μM BA with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when the embryonic axis was removed from the cotyledons and cultured on 1/2 MS medium supplement with 13.2 μM BA, 2.7 μM NAA or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA, respectively. Regenerated shoots were successfully rooted on 1/2 MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of the embryonic axis, BA, and TDZ on cotyledon regeneration was investigated in detail. Rooted plantlets were transferred to soil successfully.     

Assessment of polyembryony in lemon: rescue and in vitro culture of immature embryos

The 27 lemon cultivars analysed could be considered slightly or moderately polyembryonic, with 25 to 43% of seeds being polyembryonic and from 1.3 to 1.6 embryos per seed. On this basis, it is necessary to rescue zygotic embryos at an immature stage. Rescue and in vitro embryo development have been studied in two Citrus limon polyembryonic cultivars. Sucrose (50 and 70 g/l) was combined with Murashige and Skoog and Gamborg’s B5 media and tested for optimal growth response. An important effect of genotype was observed: embryos from cultivar ‘Eureka’ had greater survival, germination percentage, and radical development. While the sucrose concentration in the medium did not have an effect on germination, the medium affected the embryo survival and root development of the seedlings, Gamborg’s B5 medium giving the best results. The ability to form plants in vitro was affected by an increase of embryo developmental stage. The germination and seedling height were greater with embryos of seeds collected 135–150 days after anthesis.     

In vitro regeneration of plantlets from immature zygotic embryos of Picea chihuahuana Martínez, an endemic mexican endangered species

Summary Adventitious buds were induced from immature embryos of Picea chihuahuana, a species from the north of Mexico. The medium was Schenk and Hildebrandt supplemented with benzyladenine and kinetin, alone or in combination with naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid. Adventitious buds were obtained through a wide range of growth regulator concentrations, mainly from the cotyledonary region. Kinetin was more effective than benzyladenine in shoot induction. The optimum shoot induction medium contained 23.0 μM kinetin, on which 11.2 adventitious buds per embryo were obtained. Shoot development and elongation were achieved on basal SH medium at 50% concentration (SH 50) without growth regulators and improved with the reduction of sucrose concentration to 10 g l⁻¹. Differential response appeared to be associated with the provenance of the seeds, relative to the percentage of explants that responded as well as the number of adventitious buds produced per embryo. A low percentage of shoot rooting was obtained with a 24-h pulse on a medium with 16.1 or 26.85 μM of naphthaleneacetic acid. In the process of micropropagation of Picea chihuahuana, rooting is still the limiting factor.     

Ultrastructural evaluation of 8–16 cell human embryos cultured in vitro

The fine structure of eight human embryos cleaving in culture are described. Preovulatory human oocytes aspirated at diagnostic laparoscopies were fertilized and developed in vitro by methods which produced normal pregnancies. Three 8-cell and five 10–16 cell embryos showing delayed cleavage were fixed, serially sectioned and examined to determine whether the embryos were developing normally or otherwise.The organization of two 8-cell embryos was apparently normal while the other embryos showed varying aspects of abnormal development. Most blastomeres contained organelles normally present in human ova and their fine structure closely resembled those of comparable mammalian embryos. Nearly all the cellular components encountered in early mammalian embryos were observed. Certain morphogenetic changes were also noted during early cleavage involving nuclei, smooth endoplasmic reticulum and mitochondria.Both normal and abnormal features of the embryos are reported. Multi-nucleated blastomeres and partial fragmentation were commonly seen in abnormal embryos. The importance of ultrastructural evaluation of embryos in an in vitro programme is revealed in this investigation.     

High-frequency transformation from cultured cotyledons of Arabidopsis thaliana ecotypes “C24” and “Landsberg erecta”

Procedures were developed for rapid and prolific adventitious shoot regeneration of Arabidopsis thaliana (L.) Heynh ecotypes Landsberg erect and C24 from cotyledon explants at 90–100% efficiency. Immature cotyledons had the highest shoot regeneration efficiency. Prolific regeneration was achieved in Murashige and Skoog medium (MS) supplemented with 0.1–0.4 mg^–1 naphthalene acetic acid (NAA) and 1.0 mg^–1 6-benzylaminopurine (BAP) within 2 weeks. The regenerated plants had a normal phenotype and produced fertile flowers and set seeds. The above regeneration protocol was used to develop a transformation method using disarmed strains of Agrobacterium tumefaciens strains pGV3850::pH1121 based on kanamycin (Km) selection. Transgenic shoots were produced within 2–3 weeks after inoculation. Transformation of shoots was confirmed by GUS histochemical assay, as well as southern blot hybridization.Abbreviations NAA 1-Naphthalene acetic acid - BAP 6-Benzylaminopurine - Km Kanamycin - MSO Murashigea and Skoog medium [18] without hormonal supplements - GUS -Glucuronidase - NB Nutrient broth - NPT-II Neomycin phosphotransferase II - X-Gluc 5-Bromo-4-chloro-3-indolyl glucuronide - CTAB Cetyl triethylammonium bromide     

Optimization of in vitro multiple shoot clump induction and plantlet regeneration of Kentucky bluegrass (Poa pratensis)

An efficient protocol for Kentucky bluegrass (Poa pratensis L.) in vitro culture was established using shoot apices of seedlings as explants. The optimal procedure of this protocol for majority of the genotypes was that meristematic cell clumps and small calluses were firstly induced from the bases of explants on initial culture medium supplemented with 0.9 μM 2,4-d and 8.9 μM 6-BA for 20 d, then were separated and transferred to shoot clumps induction medium containing 8.9 μM 6-BA for the formation of multiple shoot clumps. The percentage of multiple shoot clumps and numbers of shoots per clump were deeply related with the combinations of different plant growth regulators, duration of initial culture, the intensity of illumination and genotypes. Histological observation of the induced explants revealed that the meristematic cell clumps were produced from repeated division of the cortical cells and original meristematic primodium cells of explants, and the multiple shoots were formed via organogenesis pathway in the meristematic cell regions of cultures on shoot clumps induction medium. In this study, plantlets were efficiently regenerated on large scale from seven cultivars of Kentucky bluegrass. Hence the meristematic cell clumps and small calluses in this protocol could be considered good targets for genetic transformation of Kentucky bluegrass.     

Phenomenon of “Siamese embryos” in cereals in vivo and in vitro: Cleavage polyembryony and fasciations

The genesis of wheat microsporial polyembryoids in vitro was analyzed in detail. The nature of different phenotypes of cereal polymeric embryos was identified. They represent the class “multiple shoot meristems,” which results from a cleavage polyembryony and is accompanied by organ fasciations of all known types (radial, flat, or ring). The morphological nature of cereal embryonic organs has been clarified: shoot meristem—axial organ; scutellum—lateral outgrowth of this axis; coleoptile—derivative of shoot meristem but fused with scutellum; terminality of scutellum—the result of linear fasciation that occurred historically. An explanation is given on how the structural model of an auxin polar transport works during the establishment of bilateral symmetry in a cereal embryo that is associated with the inverted polarization of the carrier protein PIN1 on cell membranes and, correspondingly, with the inverted auxin transport performed by this carrier (Fischer-Iglesias et al., 2001; Forestan et al., 2010).     

In vitro plantlet regeneration of “dwarf” Indian olive (Elaeocarpus robustus Roxb.): a fruit plant of Bangladesh

A plantlet regeneration protocol was developed on pot-grown mature plants of Elaeocarpus robustus Roxb. cv. Dwarf from nodal and leaf explants. The best yield of adventitious shoots was achieved from the leaf-derived calli in a modified MS (MMS₁, half strength of major salts, full strength of minor salts, and vitamins) medium containing 4.0 μM BA + 4.0 μM Kn + 0.5 μM NAA + 15% coconut water (CW). The shoot multiplication rate was amplified about twofold per culture after the addition of 15% CW to the medium. The rate of shoot multiplication reached maximum at the 5th subculture, and it maintained this rate throughout the 3 subsequent subcultures. The best rooting in vitro was investigated by subculturing the microcuttings in an MMS₂ (half strength of both major salts and minor salts and full strength of vitamins) medium containing 1.0 μM IBA in the dark for one initial week at 30°C, followed by subculturing them in a plant-growth regulator (PGR)-free medium in the light. The plantlets raised in vitro were successfully established under ex vitro conditions.     

Adventitious shoot production from strips of stem in the Dutch elm hybrid ‘Commelin’: plantlet regeneration and neomycin sensitivity

The regenerative ability of small strips of stem of the Dutch elm hybrid ‘Commelin’ was tested as well as its sensitivity to neomycins. Cambium explants (1 mm thick), were excised from woody stems collected in the field. Up to 20 buds/explant were induced within 2–3 weeks giving 2–5 rootable shoots/explant after 5–6 weeks. Shoot excision every week from week three improved the yield up to 7 shoots per explant. Fourteen and 2.9 μM GA3 promoted shoot growth. Cytokinins (1 μM zeatin or 5 μM BA or 0.05 μM TDZ) completely inhibited shoot production and promoted callus formation. Kanamycin and paromomycin at between 240 and 360 μM inhibited shoot formation as did geneticin at 80 μM. The shoot-forming ability of the explants was high from leaf fall in the autumn to the spring flush, but could be maintained up to September by using cold storage (5–7 °C). Ninety-six percent of the shoots rooted with 0.5 μM IBA and were successfully acclimatized despite having a large basal callus. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro induction, regeneration and analysis of autotetraploids derived from protoplasts and callus treated with colchicine in Citrus

In the present paper attempts were made to induce chromosome doubling of ‘Meiwa’ kumquat (Fortunella crassifolia) protoplasts and ‘Frost’ navel orange (Citrus sinensis Osbeck) embryogenic callus via colchicine treatment. Colchicine decreased protoplast viability, delayed protoplast division and inhibited callus growth, indicating presence of toxicity to cells. Cell lines established from ‘Meiwa’ protoplasts treated with 0.01 and 0.1% colchicine for 8, 16 and 24 h at each concentration showed different responses when they were cultured on embryoid-induction medium. Flow cytometry (FCM) demonstrated that tetraploids were detected in cell lines and embryoids from all of the treatments, with the highest frequency being 19.23%. As for ‘Frost’, tetraploid cells were only detected when the callus was treated with 0.1% colchicine for either 4 or 8 days, from which plantlets were regenerated. FCM and chromosome counting confirmed them as true tetraploids. The diploid cells were more active in mitotic division during a 12-day culture and smaller in size than their tetraploid counterpart. Potential applications of the novel tetraploid germplasms obtained through in vitro chromosome doubling to citrus cultivar improvement are discussed.     

Efficient in vitro regeneration of fertile plants from corm explants of Hypoxis hemerocallidea landrace Gaza—The “African Potato”

We present efficient protocols for the regeneration of fertile plants from corm explants of Hypoxis hemerocallidea Fisch. & C. A. Mey. landrace Gaza, either by direct multiple shoot formation or via shoot organogenesis from corm-derived calluses. The regeneration efficiency depended on plant growth regulator concentrations and combinations. Multiple direct shoot formation with high frequency (100% with 5–8 shoots/explant) was obtained on a basal medium (BM) supplemented with 3 mg/l kinetin (BM1). However, efficient indirect regeneration occurred when corm explants were first plated on callus induction medium (BM2) with high kinetin (3 mg/l) and naphthalene acetic acid (NAA 1 mg/l), and then transferred to shoot inducing medium (BM3) containing BA (1.5 mg/l) and NAA (0.5 mg/l). Shoot regeneration frequency was 100% and 30–35 shoots per explant were obtained. The regenerated shoots were rooted on a root inducing medium (BM4) containing NAA (0.1 mg/l). Rooted plantlets were transferred to the greenhouse. The regenerants were morphologically normal and fertile. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. Efficient cloning protocols described here, have the potential not only to substantially reduce the pressure on natural populations but also for wider biotechnological applications of Hypoxis hemerocallidea—an endangered medicinal plant.     

Induction of Haploid Morphogenic Calluses from in vitro Cultured Anthers of Prunus Armeniaca cv. ‘Harcot’

Apricot (Prunus armeniaca) ‘Harcot’ anthers, were cultured in vitro for the production of haploid plants. The best androgenic response was achieved with Nitsch and Nitsch (1969) medium, supplemented with 4.52 μM 2,4-D, 4.52 μM zeatin, 2.85 μM IAA and 40 g l⁻¹ sucrose. Cultures were maintained in the dark for 8 days, at 28°C, followed by transfer to a 16-h photoperiod, with 35 μm m⁻² s⁻¹ light intensity and 24/22°C day/night temperature. The androgenic response was correlated with the floral bud size, its phenologic stage and the level of microspore evolution. Anthers containing microspores at the tetrad/uninucleate stage were the most appropriate. The ploidy level of the calluses was evaluated by flow cytometry revealing that they range from haploid to octaploid. Mixoploid calluses have also been identified. Histological studies showed that the haploid calluses have their origin in the microspores. Nodular structures consisting of cells with dense cytoplasm and differentiated xylem elements were observed and were surrounded by an autofluorescent layer, probably due to cutin deposition.     

Methylation patterns in 5＇ terminal regions of pluripotency-related genes in bovine in vitro fertilized and cloned embryos

In order to investigate DNA methylation profiles of five pluripotency-related genes (Oct4, Sox2, Nanog, Rex1 and Fgf4) during bovine maternal to zygotic transition (MZT) in both in vitro fertilized (IVF) and nuclear transfer (NT) embryos, sodium bisulfite sequencing method was used to detect DNA methylation levels, accompanied by the statistical analysis of embryo developmental rates. The results showed that Oct4, Nanog, Rex1 and Fgf4 were respectively demethylated by 25.22% (P < 0.01), 3.84% (P > 0.05), 31.82% (P < 0.01) and 10% (P > 0.05) while Sox2 retained unmethylation during MZT in IVF embryos. By contrast, Oct4 and Rex1 respectively underwent demethylation by 23.04% (P < 0.01) and 6.02% (P > 0.05), and, reversely, Sox2, Nanog and Fgf4 respectively experienced remethylation by 0.84% (P > 0.05), 5.39% (P > 0.05) and 5.46% (P > 0.05) during MZT in NT embryos. Interestingly, the CpG 14 site of Sox2 was specifically methylated in both 8-cell and morula NT embryos. In addition, the development of blastocysts between IVF and NT embryos showed no significant difference. DNA methylation analysis showed that only Oct4 and Sox2 underwent the correct methylation reprogramming process, which may be responsible for the development of blastocysts of NT embryos to a certain extent. In conclusion, the five genes respectively experienced demethylation to different extents and incomplete DNA methylation reprogramming during bovine MZT in both IVF and NT embryos, suggesting that they may be used as indicators for bovine embryo developmental competence.     

Plant regeneration from hypocotyls,cotyledons and stem segments of the C3–C4 intermediate species Moricandia arvensis

Techniques have been developed for the regeneration of Moricandia arvensis from complex explants. Hypocotyl segments and cotyledonary explants regenerated shoots, but the most efficient plant regeneration was from stem sections taken from in vitro grown shoots. Regeneration from these three explant types was tested on a range of concentrations of benzylaminopurine and either naphthylene acetic acid or indole acetic acid. Regeneration from all three explants was much higher on indole acetic acid than on naphthylene acetic acid and the ratio of auxin to cytokinin was also significant in determining the response of explants. Optimum regeneration was on 1mg/l IAA with 1mg/l BAP. Plants could be transferred to soil and grown to flowering in the glasshouse.Abbreviations GDC glycine decarboxylase - BAP benzyl aminopurine - NAA naphthalene acetic acid - IAA indole acetic acid     

Plantlet regeneration from mature zygotic embryos and embryonic explants of masson pine （Pinus massoniana Lamb.）

Excised zygotic embryos,cotyledons and hypocotyls of juvenile seedlings of masson pine were grown on DCR medium supplemented with several concentrations of various plant phytohormones.BA(1.0mg/L) in combination with NAA(0.05mg/L) in DCR medium was found to increase the formation of adventitious buds from mature zygotic embryos,but most of them were formed at the tips of embryonic cotyledons.Adventitious buds were obtained from cotyledons and hypocotyls from juvenile seedlings when they were cultured on DCR medium containing BA 3-5 mg/L and NAA 0.1-0.2 mg/L.Elongation of buds were observed on hormone-free DCR medium with or without activated charcoal(0.5%).Root initiation was achieved with full or half strength DCR medium supplemented with IBA 1.0 mg/L and NAA 0.25-0.5 mg/L.Approximately 11-20 axillary buds formed on each explant when juvenile seedling explants were treated(3-20h) with BA 50-100 mg/L,followed by transfer to hormone-free DCR medium.The maximum number of shoots obtained per explant within six months was 33.     

Nutrient requirements of in vitro cultured Halophila ovalis and Posidonia coriacea: nitrogen source

Axenic cultures of Posidonia coriacea and Halophila ovalis were established to investigate the effect of nitrogen addition and the optimum concentration of MS (Murashige and Skoog Physiol Plant 15:473–497, 1962) macronutrients, micronutrients and organics. Chlorophyll content and dissolved oxygen of H. ovalis was optimal at the control concentration of macronutrients (half strength MS). Below and above this concentration, growth of this species was compromised. A similar effect was observed for H. ovalis growth in response to MS micronutrient. However, changing the MS vitamins concentration had no impact on the growth of H. ovalis or P. coriacea. Leaf and root numbers of H. ovalis were highest in the treatment with the most nitrogen (50.6 mM). Root architecture of H. ovalis was affected by nitrogen source; shorter roots without root hairs were produced when NH₄NO₃ was present in the media. Longer roots with root hairs were produced when the cultures were grown in the absence of NH₄NO₃. P. coriacea growth did not exceed that of the control for any of the treatments. The response of H. ovalis to nutrient addition suggests its opportunistic nature, whereas the lack of response by P. coriacea suggests there may be some other limiting factor. This study shows that the proposed formulation of ½ strength MS can be suitable for both H. ovalis and P. coriacea.     

The ability of Prunus avium× P. pseudocerasus `Colt' to form somatic embryos in vitro contrasts with the recalcitrance of P. avium

Somatic embryos were induced on roots excised from in vitro plants of Prunus avium× pseudocerasus `Colt'. On medium containing 6-benzylamino purine (BAP, 1.5 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D, 15 μM), a mean of 25 (s.e. ± 2.0) somatic embryos were produced on intact root systems and 15 (s.e. ± 1.7) on roots systems cut into 10 mm pieces. Most somatic embryos were formed directly on intact roots and indirectly (from callus) on sectioned roots. A mean of 2.5 (s.e. ± 0.25) secondary embryos per primary embryo were formed directly on primary embryos after they were transferred to medium containing BAP (1.5 μM), indole-3-butyric acid (10 μM) and 2,4-D (5 μM). After transfer to a medium containing BAP (2 μM) and gibberellic acid (GA<sub>3</sub>, 3 μM), shoots developed in 75% (s.e. ± 7.3) of the embryos. Somatic embryos were not induced on explants of in vitro roots or shoots of P. avium, and were induced infrequently on zygotic embryos, although a wide range of media were tested. Possible reasons for the contrasting embryogenic ability of `Colt' and P. avium are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic variation at storage protein-coding loci of common wheat (cv ‘Chinese Spring’) induced by nitrosoethylurea and by the cultivation of immature embryos in vitro

Electrophoretic patterns of seed storage proteins, the high-molecular-weight glutenins and gliadins, were studied in 468 plants of the common wheat cultivar Chinese Spring regenerated from callus culture of immature embryos, in 115 plants grown from seeds treated with nitrosoethylurea and in 260 control plants. From 5 to 21 single grains were analysed from each plant. In these three groups, the frequency of inherited mutations causing the loss of all proteins controlled by a locus (null-mutations, probably caused by a chromosomal deficiency) was 0.69%, 2.07%, and 0.05% per locus (the differences were statistically significant), respectively, while that of mutations causing the loss of a single protein band was 0.11%, 0.33%, and 0.05%, respectively. The loss of all of the gliadins controlled by Gli-B1 or GH-B2 (mutations were probably caused by a deletion of satellites of the corresponding chromosomes), was significantly higher than the loss of gliadins controlled by genomes A and D. Gene mutations altering the electrophoretic mobility of a single protein band in the pattern were found only in the second group of plants (0.44%). Therefore, chemical mutagenesis which produced not only more mutations than cultivation of immature wheat embryos in vitro, but also a higher ratio of mutations that altered DNA sequences, can be considered as an easier and comparatively more promising way for obtaining new improved variants of loci controlling biochemical characteristics in wheat. Somaclonal variation, on the other hand, was probably mainly caused by chromosomal abnormalities and could therefore hardly be considered as a useful tool in wheat breeding.