Embryogenesis and plant regeneration from callus cultures derived from unpollinated ovaries of Hyoscyamus muticus L.

Unpollinated ovaries of Hyoscyamus muticus L. (commonly known as Egyptian henbane) were cultured on Murashige and Skoog and Bourgin and Nitsch media supplemented with various growth hormones to study the organogenesis, embryogenesis and regeneration of plantlets. Embryogenesis was reported for callus grown on both media containing 0.05 mg/l α-naphthaleneacetic acid and 0.5 mg/l 6-benzylaminopurine. Differentiation of roots and shoots from the calli also occurred in these media. Albinism or chlorophyll deficiency and variation in ploidy level were observed among the ovary-derived plantlets. Received: 7 April 1997 / Revised received: 2 August 1997 / Accepted: 2 September 1997     

Ploidy variation of pronamide-treated maize calli during long term culture

Summary Anther-derived calli of corn were treated with 10 M pronamide for 2, 3 and 4 days. The ploidy level of the calli was then evaluated using flow cytometry, at different times after the treatment. Untreated haploid calli did not change in ploidy level for 97 days but by 466 days, there were up to 50% diploid or higher ploidy cells thus showing that spontaneous doubling may occur during corn calli subculture with this genotype. Pronamide treatment did increase the percentage of diploid and tetraploid cells and by 466 days, all of the lines showed an additional change toward higher ploidy levels. This change may be due to spontaneous chromosome doubling or to differential cell cycle times of cells with different ploidy levels. The ploidy level of plants regenerated from the cultures was determined by counting the guard cell chloroplast numbers and the correlation with the ploidy level of the cultures was r²=0.84. These studies show that pronamide treatments can increase haploid maize callus chromosome numbers and that spontaneous chromosome doubling can occur with time in maize callus.     

尾巨桉胚状体诱导及愈伤组织差异研究

以尾巨桉优良无性系无菌苗茎段为外植体,通过对多种不同浓度生长调节剂组合的优化,进行胚状体诱导研究;并对胚性与非胚性愈伤组织进行形态解剖学观察、相关生理指标检测以及相关基因荧光定量PCR分析,以揭示尾巨桉胚性愈伤组织非胚性化发生的机理,为建立尾巨桉体细胞胚胎再生体系提供参考。结果表明:(1)胚性愈伤组织在MS+0.1mg/L NAA+0.01mg/L TDZ培养基中诱导得到胚状体,外植体经过0.5mol/L蔗糖处理12h有助于胚性愈伤组织产生胚状体,胚状体最高发生率为16.7%。(2)尾巨桉胚性与非胚性愈伤组织石蜡切片观察发现,两者的细胞形态特征存在明显的差异,胚性愈伤组织细胞体积小,排列紧密,表现出典型的胚性细胞特征,而非胚性细胞比较大,排列疏松,细胞呈不规则形状。(3)生理生化指标检测结果表明,非胚性愈伤组织中蛋白质含量、SOD、PPO及CAT活性均显著低于胚性愈伤组织,非胚性愈伤组织中木质素、可溶性糖含量以及PAL和POD活性要高于胚性愈伤组织,二者的反肉桂酸4-单加氧酶基因、淀粉磷酸化酶基因、谷胱甘肽硫转移酶基因、葡萄糖-1-磷酸腺苷酸转移酶基因、葡萄糖六磷酸异构酶基因、分支酸合酶基因以及苯丙氨酸解氨酶基因表达差异也达到显著水平。     

The role of irradiation dose and DNA content of somatic hybrid calli in producing asymmetric plants between an interspecific tomato hybrid and eggplant

Highly asymmetric somatic hybrid plants were obtained by PEG/DMSO fusion of gamma-irradiated mesophyll protoplasts of the kanamycin-resistant (KmR⁺) interspecific hybrid Lycopersicon esculentum x L. pennellii (EP) with mesophyll protoplasts of Solanum melongena (eggplant, E). Elimination of the EP chromosomes was obtained by irradiating the donor genome with different doses of gamma rays (100, 250, 500, 750 and 1000 Gy). The selection of somatic hybrid calli was based on kanamycin resistance; EP and E protoplasts did not divide due to the irradiation treatment and sensitivity to kanamycin, respectively. KmR⁺ calli were recovered following all irradiation doses of donor EP protoplasts. The hybrid nature of the recovered calli was confirmed by PCR amplification of the NptII gene, RAPD patterns and Southern hybridizations using potato ribosomal DNA and pTHG2 probes. Ploidy levels of calli confirmed as hybrid were further analyzed by flow cytometry. Such analyses revealed that the vast majority of hybrid calli that did not regenerate shoots were 5–9n polyploids. The three asymmetric somatic hybrid plants obtained were regenerated only from callus with a ploidy level close to 4n, and such calli occurred only when the donor EP had been exposed to 100 Gy. The amount of DNA in somatic hybrid calli, from 100-Gy exposure, was found by dot blot hybridization with the species-specific probe, pTHG2, to be equivalent with 3.1–25.8% of the tomato genome. Thus, DNA contained in 3.8–13.2 average-size tomato chromosomes was present in these hybrid calli. The asymmetric somatic hybrid plants had the eggplant morphology and were regenerated from one hybrid callus that contained an amount of tomato DNA equivalent to 6.29 average-size tomato chromosomes.     

Vertical heterogeneity of DNA ploidy level assessed by flow cytometry in calli of Passiflora Cincinnata

During in vitro culture conditions, callus tissue is exposed to different intensities of environmental stress, which may induce somaclonal variation. Among the possible resulting abnormalities, callus cells can exhibit distinct DNA ploidy levels, a type of somaclonal variation associated with euploidy and/or aneuploidy. As somaclonal variation has been regarded as both a positive and negative phenomenon, the development of strategies to carefully assess the stability of DNA ploidy level within callus tissue is highly valuable. To this end, the present work aimed to evaluate the presence of intra- and inter-calli heterogeneity in relation to DNA ploidy level and nuclei density by flow cytometry. Calli were induced from cotyledonary leaves of Passiflora cincinnata, a wild passion fruit species. Embryogenic friable calli cultivated for 2, 6, and 9 mo were classified as young, intermediary, and old, respectively. These calli were horizontally sliced from the bottom-up at approximately the same thickness, and a total of 160 layers were evaluated by flow cytometry. Inter- and intra-calli heterogeneities were detected in relation to nuclei density and DNA ploidy level. Additional analysis was performed to identify the most proliferative layer. We conclude that care must be taken when using callus as source material for flow cytometry, since one portion cannot represent the whole cell mass. Moreover, in order to prevent the emergence of undesired ploidies during clonal propagation, callus culture time should not be prolonged.     

Asymmetric protoplast fusions between wild species and breeding lines of potato – effect of recipients and genome stability

 The objective of this study was to investigate if in asymmetric protoplast fusion experiments the ploidy of the recipient line (di-haploid and tetraploid) has an influence on the extent of the asymmetry of the regenerating fusion products. Nineteen different experiments with the wild species Solanum bulbocastanum and Solanum circaeifolium as donors (irradiated with 210 Gy) and different breeding lines (di-haploid and tetraploid) were carried out. The degree of genome elimination was determined by measuring the relative DNA content using flow cytometry. The data showed that the loss of DNA in hybrid plants was significantly higher for 4x, compared to 2x, plants as recipients. In addition, the stability of asymmetry in the fusion products was studied. For this purpose differences in asymmetry in individual shoots originating from the same callus were analysed. A large variation in the DNA content of individual shoots was detected. Of the 4x to 6x shoots 44% had the same DNA content as another shoot originating from the same callus, 19% had a DNA content between 4x and 6x but different from any other analysed shoot originating from the same callus, 2% were chimeras and 35% had a completely different DNA content (eutetraploid, euhexaploid, eupolyploid or asymmetric with a ploidy level above 6x). RFLP-analysis with single-copy probes of 12 regenerates from six calli (two regenerates per callus) confirmed the assumption that the different regenerates of one callus originate from the same single cell. The analysis of selected regenerates cultivated for a period of more than 1 year demonstrated that the genome of asymmetric regenerates might change during cultivation. Received: 30 April 1998 / Accepted: 24 July 1998     

Establishment of stable NaCl-resistant rice plant lines from anther culture: distribution pattern of K+/Na+ in callus and plant cells

 Soil salinity markedly suppresses the growth of rice (Oryza sativa L.). We established rice anther culture to select for rice callus lines adapted to NaCl stress and regenerated plant progenies resistant to a NaCl stress of E.C. 16–18 mS. When exposed to NaCl, NaCl-adapted rice calli lost K⁺ and accumulated little Na⁺. Conversely, plant cells lost relatively little K⁺ and accumulated Na⁺. It is plausible that, NaCl-resistant mechanisms are different at callus and plant level. The stable NaCl-resistant lines produced have potential use in elucidating the molecular mechanisms behind NaCl resistance in rice and in rice breeding. Received: 27 February 1997 / Accepted: 4 April 1997     

In vitro plant regeneration from callus of niger (Guizotia abyssinica Cass.) cv. Sahyadri

Callus formation was achieved with root, hypocotyl, and cotyledon explants of niger (Guizotia abyssinica Cass.) cultivar Sahyadri on Murashige and Skoog medium containing 0.5 mg l^–1 β-indoleacetic acid + 1.5 mg l^–1 6-benzylaminopurine (BAP). Hypocotyl and cotyledon-derived calli when transferred onto a medium with 0.5 mg l^–1 BAP produced an average of 12–32 shoots/ callus culture. The callus retained its potential for shoot regeneration for more than 19 months. The shoots formed an extensive root system and were transferred to pots kept in a greenhouse, where the survival rate was 98%. The plantlets flowered in vitro if transfer to fresh medium or to soil was delayed by 40–50 days. All regenerants were diploid with 2n=30. Received: 13 March 1997 / Revision received: 17 May 1997 / Accepted: 5 July 1997     

Efficient plantlet regeneration from protoplasts isolated from suspension cultures of poplar (Populus alba L.)

We developed an efficient plant regeneration system from protoplasts for poplar (Populus alba L.). Protoplasts were isolated from 4-day-old suspension cultures derived from seed-induced calli with a yield of 6.96× 10⁶ cells/g fresh weight cells and then cultured at a concentration of 2.5×10⁵ cells/ml in NH₄NO₃-free Murashige and Skoog (MS) medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 μM thidiazuron (TDZ) and 0.5 M glucose as a osmoticum. The plating efficiency of the cultured protoplasts was calculated at 26.5% at day 7 and 31.7% at day 14. Cell colonies were observed after culturing for 4 weeks. Regenerated colonies were propagated through subculture in liquid MS medium supplemented with 5 μM 2,4-D. Buds were induced from regenerated calli on MS medium containing 10 μM kinetin or 1 μM TDZ. Regenerated shoots were rooted on half-strength MS medium, and the plantlets were transplanted in soil. Randomly amplified polymorphic DNA analysis did not detect any DNA polymorphism among the regenerated plants. Received: 7 March 1997 / Revision received: 16 June 1997 / Accepted: 5 July 1997     

Flow cytometric analyses in embryogenic and non-embryogenic callus lines of Cyclamen persicum Mill.: relation between ploidy level and competence for somatic embryogenesis

Embryogenic and non-embryogenic callus lines derived from the same diploid Cyclamen persicum genotype (`Purple Flamed') were analyzed by flow cytometry and compared to the initial plant material. The DNA content of the diploid plant in the greenhouse was 1.12 pg DNA/2C as estimated in relation to the internal standards tomato nuclei and chicken erythrocytes. In both callus lines the majority of cells contained the same amount of DNA as the initial plant, indicating that no polyploidization has taken place after 5 years of culture on medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 mg/l 6-(γ-γ-dimethylallylamino)purine(zip). Thus, our data suggest that in Cyclamen callus lines there was no strict correlation between the ploidy level and the ability to produce somatic embryos. Furthermore, following the proportion of cells in the three phases of the cell cycle (G0/G1, S, G2/M) during one subculture period of 4 weeks revealed high division activity within the first 2 weeks for both callus lines cultured on the 2,4-D-containing medium. However, when transferred to hormone-free medium, the division activity of the embryogenic cell line decreased markedly, corresponding to the differentiation of somatic embryos. In contrast, for the non-embryogenic callus an increase in cells in the G2/M phase was observed. Received: 22 November 1996 / Revision received: 6 January 1997 / Accepted: 20 February 1997     

Chromosome doubling of the bioenergy crop,Miscanthus×giganteus

The perennial grass, Miscanthus×giganteus is a sterile triploid, which due to its growth rate and biomass accumulation has significant economic potential as a new bioenergy crop. The sterility associated with the triploid genome of this accession requires labor‐intensive vegetative, instead of seed propagation for potential commercial production. Chromosome doubling was used to produce hexaploid plants in an effort to restore fertility to M×giganteus. Tissue culture derived calli from immature inflorescences were treated with the antimitotic agents, colchicine and oryzalin in liquid and solid media. Calli survival rate decreased with increasing concentrations and durations of colchicine or oryzalin treatments and ranged from 0% to 100%. Nuclear DNA content, as determined by flow cytometry, indicated that the frequency of chromosome‐doubled calli varied between compounds and concentrations with the greatest proportion of callus doubling observed using 2‐day treatments of 15 μm oryzalin (78%) or 939 μm colchicine (67%). Liquid media treatments were more effective than solid gels for chromosome doubling. Although oryzalin was effective at chromosome doubling, it inhibited callus growth and plant regeneration frequency. Seven hexaploid plants with doubled DNA content were generated, which displayed increased stomata size (30.0±0.2 μm) compared with regenerated triploid M. ×giganteus plants (24.3±1.0 μm). Following clonal replication these plants will be evaluated for growth rate, biomass accumulation, and pollen viability. Successful chromosome doubling and plant regeneration of M.×giganteus suggests that ploidy manipulation of this plant and its parental species (Miscanthus sinensis and Miscanthus sacchariflorus) could be a means to access genetic variability for the improvement of Miscanthus as a biofuel/bioenergy crop.     

High Uniformity of plants regenerated from cytogenetically variable embryogenic suspension cultures of poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch)

Summary Shoot tip explants, callus and embryogenic cell suspensions of Euphorbia pulcherrima have been examined for quantitative variation in nuclear DNA content by means of cytophotometry. Increasing instability was found in calli and cell suspensions from Erlenmeyer flask and bioreactor culture. Nuclear DNA content ranged from 2C up to 32 C. Plants regenerated from embryogenic cell suspensions, however, were highly uniform with regard to phenotype and ploidy level indicating strongly impaired embryogenic potential of polyploid, aneuploid or other genetically altered cells.     

Friable embryogenic callus and somatic embryo formation from cotyledon explants of African marigold (Tagetes erecta L.)

Embryogenic callus and somatic embryos were induced from cotyledonary explants of African marigold (Tagetes erecta L.). Cotyledons were first cultured on MS medium supplemented with 2.0 mg l^–1 2,4-D and 0.2 mg l^–1 kinetin. After 5 weeks, calli were transferred to MS medium supplemented with 0.02 mg l^–1 thidiazuron where compact embryogenic callus developed. Friable embryogenic callus developed when the compact embryogenic callus was transferred to medium containing 2,4-D and subcultured every 2 weeks. Friable embryogenic callus has been maintained for more than 2 years without losing the capacity to generate embryos. Embryo development was obtained when friable embryogenic callus was transferred to MS medium supplemented with 3 mg l^–1 ABA and 60 g l^–1 sucrose. The addition of 10–30 mM l-glutamine improved embryo development. Received: 13 May 1997 / Revision received: 24 February 1998 / Accepted: 28 March 1998     

Organogenesis and plant regeneration in Zephyranthes rosea Lindl.: Histological and chromosomal study

The genus Zephyranthes rosea is a member of the family Amaryllidaceae. The plant is widely cultivated as ornamental. The objective of this study was to optimize an in vitro propagation method for the production of genetically stable Z. rosea plant. The chromosomal status of the regenerated plants was also studied to determine their ploidy levels and to identify the structural and numerical variations, if any. Two explants of Zephyranthes rosea, i.e. bulb scale and flower bud (3–4 mm each), were used and incubated in a culture room at 25 ± 2°C in which two different types of calli were induced from two sources. The MS medium amended with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5–2.0 mg/l) successfully induced callus from bulb-scale explants (50.25–57.5%). The addition of coconut water (10%) in 2,4-D-added medium further improved the callus induction frequency (68.4%). Bulb-scale calli were found to be highly regenerative while flower-bud calli did not show any organogenetic responses. The use of plant growth regulators, such as naphthaleneacetic acid (NAA) + benzylaminopurine (BAP), was found to be very effective for shoot bud development; maximum shoot number (11.50/callus mass) was observed in NAA (0.5 mg/l) + BAP (1.0 mg/l) added medium. Histological analysis of callus revealed that the origin of the shoot bud was de novo. Rooting frequency (65.25%) and the number of roots (7.5/shoot) were best achieved in indole-3-butyric acid (4.0 mg/l)-amended medium, followed by indole-3-acetic acid (4.0 mg/l). The regenerated Z. rosea plants showed 2n = 24 chromosome numbers.     

Chemical and genetic characterization of calli derived from somatic hybridization between tansy (Tanacetum vulgare L.) and pyrethrum (Tanacetum cinerariifolium (Trevir.) Schultz-Bip.)

 Pyrethrum (Tanacetum cinerariifolium (Trevir.) Schultz-Bip.) produces environmentally benign pesticides, the pyrethrins, and tansy (Tanacetum vulgare L.) lower terpenes of variable biological effectiveness. As an approach to improve the oil content and composition of tansy for enhanced biological activity, a somatic hybridization technique between tansy and pyrethrum was established. About 1×10⁶ of leaf-mesophyll protoplasts of both species were mixed and fused with a solution containing 15% polyethylene glycol. Light-green and yellowish calli developed from the fusion experiments. The fusion-derived calli grew vigorously on MS medium supplemented with 6.4 mg l^-1 of BAP, 0.8 mg l^-1 of NAA, and 30–40 g l^-1 of glucose. Nuclear DNA content, RAPD patterns, and volatile compounds were analyzed to determine the hybridity of the calli. The nuclear DNA content of the tansy and pyrethrum genotypes, and the protoplast-derived calli of tansy were 6.41, 7.39, 13.84, and 8.11 pg, respectively. The nuclear DNA content of individual calli derived from the protoplast fusion between tansy +tansy ranged from 8.84 (F43A) to 19.59 pg (F43C) while those of the tansy+pyrethrum fusions were 10.66 (F46A) and 31.87 pg (F46B). Using four 10-mer primers a total of 56 RAPD-PCR fragments were produced. The distance matrices of fragments were calculated by average linkage cluster analysis. Two visually separated clusters were observed. One cluster consisted of the two tansy genotypes and the fusion-derived callus F43A; the other consisted of pyrethrum and fusion-derived calli F46B and F46C. Volatile compounds, such as decadienal, artedouglasia oxide, heptadecane, syringaldehyde and coniferyl alcohol, analyzed by gas chromatography mass spectrometry, were found only in the protoplast fusion-derived calli F43A and F46B. Several less volatile compounds were also detected only in fusion calli. Hexadecanoic and linoleic acids were common to fusion-derived calli and tansy, and one unknown compound to fusion-derived calli and pyrethrum. Pyrethrins I and II were detected from pyrethrum, but not from the fusion-derived calli. The additive nuclear DNA content of protoplast fusion-derived calli and the results of the RAPDs suggest that interspecific fusions had occurred. The small number of volatile compounds detected from both the fusion calli and from the donor species indicates that the unorganized callus tissue is unable to produce tissue-specific volatile compounds. Received: 4 August 1998 / Accepted: 30 September 1998     

Effect of cadmium on the phenolic compounds formation in the callus cultures derived from various organs of the tea plant

The effects of cadmium (6.3 × 10^?5 M or 10.6 × 10^?5 M) on the growth of tea plant (Camellia sinensis L.) callus cultures derived from leaves, stems, and roots and on the formation, in these cultures, of phenolic compounds, including flavans and lignin, which are characteristic of the tea plant, were investigated. In the calli derived from leaves and stems, cadmium treatment decreased the biomass increment, while in the calli derived from roots, growth characteristics remained at the control level. Under the effect of cadmium, the content of phenolic compounds, including flavans, in the leaf calli decreased, while in the stem and root calli, it either increased (at the cadmium concentration of 6.3 × 10^?5 M), or was close to a control one (at the cadmium concentration of 10.6 × 10^?5 M). The lignin content in the root and stem calli increased, but it did not change in the leaf calli. All this data demonstrate that the cadmium-induced changes in phenolic metabolism of the tea plant callus culture depended both on the cadmium concentration in the medium and on the origin of calli.     

Flow cytometric analysis of somatic embryos, shoots, and calli of the cactus Copiapoa tenuissima Ritt. forma monstruosa

Flow cytometry was used for analyzing DNA contents of nuclei isolated from in vitro grown somatic embryos, shoots and calli, as well as mammillae of in vivo grown shoots of cactus, Copiapoa tenuissima Ritt. forma monstruosa. Endoreduplication was detected in both in vitro grown somatic embryos, shoots and calli and in mammillae derived from in vivo grown shoots. However, the lowest ploidy levels ranged from 2C to 4C in somatic embryos, and reaching up to 32C for in vitro grown shoots and calli from mammillae. Whereas, ploidy levels of in vivo-derived mammillae ranged from 2C to 16C. The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium had no influence on levels of ploidy of regenerated tissues. The mean genome size of cactus was calculated as 2C = 2.87 ± 0.05 picogram (pg).     

Ploidy levels in Beta vulgaris (red beet) plant organs and in vitro systems

The ploidy levels of the cells in different organs (leaves, petioles and roots) of red beet (Beta vulgaris L.) plants of different ages, as well as of different in vitro systems (transformed hairy roots, calli derived from leaves and rhizogenic calli), were investigated using flow cytometry. Two callus lines with red and yellow phenotypes, derived by mechanical separation of the morphologically heterogeneous rhizogenic callus, were also examined. All investigated samples experienced several cycles of endoreduplication. The older organs exhibited higher levels of polysomaty than the young ones. The highest degree of endoreduplication was found in old petiole tissue and the lowest in the red callus line (cycle values of 1.81 and 0.55, respectively). Interestingly, the callus derived from leaves did not exhibit a 2Cx peak, but was tetraploid, probably due to genetic instability, which may have been caused by prolonged cultivation under in vitro conditions. Red and yellow calli showed significantly lower polysomaty (cycle values of 0.55 and 0.59, respectively) than the primary rhizogenic callus (cycle value of 1.09). The DNA profiles of the two phenotypes differed, possibly reflecting differences in their metabolism.     

Somatic embryogenesis and plant regeneration in Lilium longiflorum Thunb

Friable callus was obtained from styles and flower pedicels of Lilium longiflorum Snow Queen and the Oriental lily hybrid Star Gazer on Murashige and Skoog (MS) media containing either 2 μm dicamba or 2 μm picloram. Cell suspension cultures were established by suspending the callus of L. longiflorum Snow Queen in liquid medium containing 2 μm dicamba. Through a purification process, a fine fast-growing cell suspension was obtained. This suspension was composed of a homogenous population of small dense cells, which tended to organise into embryo like structures (ELS). In liquid culture with the auxin dicamba, the ELS underwent continuous callus formation. When transferred to solidified hormone-free MS medium, the ELS germinated, forming complete plantlets. Histological investigation showed that in the ELS both shoot and root meristems were distinctly evident. It was concluded that the ELS obtained were in fact somatic embryos. Received: 4 April 1997 / Revision received: 13 May 1997 / Accepted: 15 June 1997     

Callus induction and somatic embryogenesis of Phalaenopsis

Callus induction and plant regeneration through somatic embryogenesis in Phalaenopsis Richard Shaffer `Santa Cruz' were examined. Protocorm-like body (PLB) segments formed calli in Vacin and Went medium with sucrose. The optimal concentration of sucrose was 40 g ⋅ l^–1. Medium containing 200 ml ⋅ l^–1 coconut water together with 40 g ⋅ l^–1 sucrose was effective for callus induction. Gellan gum was suitable than agar as a gelling agent for callus induction. The calli easily formed PLBs after being transferred to a medium without sucrose. Histological observation suggested that the PLBs were somatic embryos. No variation was observed in the flowering plants regenerated through somatic embryogenesis. Received: 11 June 1997 / Revision received: 6 October 1997 / Accepted: 18 October 1997