Androgenesis in vitro in anther cultures of chlorophyll mutants ofNicotiana tabacum

The course of androgenesisin vitro was investigated with anther cultures of two chlorophyll mutants of Nicotiana tabacum. A different sensitivity to the hormonal composition of the medium was revealed between the cultures White Seedling and Sulfur; the stimulatory effect of kinetin on the frequency of androgenesis was observed only in White Seedling cultures. In addition to green plants, “aurea” (mutation Sulfur) or “albino” (mutation White Seedling) phenotypes also differentiated in both cultures. The possible causes of variability in the participation of green and mutant forms are discussed.     

Isoperoxidase patterns in leaf tissues of a genome series of two cultivars ofNicotiana tabacum L

Polyacrylamide gel electrophoresis was used to study the differences in patterns of the isoperoxidase spectrum in leaf tissues of the genome series (2n, 3n, 4n) of twoNicotiana tabacum L. cultivars,i.e. in the growth stage of the 5th–6th and 10th–11th leaf, and in the stage of elongation growth. Mutual comparisons of the cultivars showed that the variability and difference in patterns between the cultivars in later growth stages was more important. In both cultivars only one rapidly migrating main band of isoenzymes was registered within the range of Rm 0.45–0.77; only in the stage of elongation growth on the 2n and 4n levels two bands were recorded. No association between the number of genomes and the number or distribution of isoperoxidases was found.     

Organization of actin filaments in regenerating and outgrowing subprotoplasts from pollen tubes ofNicotiana tabacum L.

The dynamics of actin-filament organization in pollen-tube subprotoplasts ofNicotiana tabacum L. cv. Samsun during regeneration and outgrowth was examined using phalloidin probes and a non-fixation method. A succession of actin arrays was examined during subprotoplast regeneration that strongly resembled the actin dynamics described for developing microspores by Van Lammeren et al. (1989, Planta178, 531–539) and activated pollen by Tiwari and Polito (1988, Protoplasma147, 5–15). At the end of the succession the actin filaments often became extended between two opposite polar foci. The ordering of the cortical actin filaments reflected a polarity in the subprotoplasts which determined the plane of outgrowth. The site of outgrowth was often marked by a ring of actin filaments. As growth proceeded and tube-like structures were formed, the arrangement of cortical actin filaments was found to be transverse to the elongation axis. Since the patterns of actin distribution were identical in both caryoplasts and cytoplasts, it was concluded that the pollen-tube cytoplasm has the intrinsic capacity of reorganizing actin filaments and imposing polarity on the spherical subprotoplasts.     

Regeneration of plants from leaf explants of Cucumis melo cv. Pusa Sharbati

Leaves of three different sizes excised from 14, 21, 28 and 35-day-old seedlings of Cucumis melo were cultured on a MS medium supplemented with a range and combination of growth regulators. Maximum shoot differentiation from the leaf explants occurred in the combined presence of BAP and 2iP at equimolar concentration of 1 M. Regeneration potential of leaves declined with increasing size of the leaves and the age of the donor seedlings. For elongation the shoots were transferred to MS+BAP [1 M]. Such shoots were rooted with 75% frequency on MS+IAA [0.5 M]. The plants have been established in pots.Abbreviations BM Basal Medium - MS Murashige and Skoog - BAP 6-Benzyl amino-purine - 2iP 6- V, V -dimethylallylamino purine - IAA Indole-3-acetic acid     

Adventitious shoot regeneration from leaf explants of Gypsophila paniculata L.

Adventitious shoots were successfully regenerated from leaf explants of Gypsophila paniculata L. The efficiency of shoot regeneration for cv. Arbel was tested on 18 media based on Murashige and Skoog basal medium containing different concentrations of thidiazuron or 6-benzylaminopurine in combination with naphthaleneacetic acid. Both explant age and that of the cuttings used as leaf donors affected the regeneration efficiency. The highest efficiency of adventitious shoot regeneration was obtained with the oldest leaves originating from the youngest cutting analyzed; on thidiazuron-containing medium, shoots regenerated on average from 67% of the leaves, with an average of seven shoots per explant. This regeneration procedure was suitable for all six commercial cultivars studied. Regenerated shoots elongated, rooted and successfully acclimatized to the greenhouse where they were grown to flowering. Received: 25 July 1998 / Revision received: 11 November 1996 / Accepted: 30 November 1996     

Direct flower neoformation from superficial tissue of small explants of Nicotiana tabacum L.

Summary On explants composed of 3–6 layers of epidermal and sub-epidermal cells of Nicotiana tabacum L. from the floral branches, it is possible to obtain mitoses followed very rapidly by meioses and the direct formation of anthers and pistil without any intermediate callus.     

Regeneration of plants from leaf explants of micropropagated clonal Eucalyptus grandis

Summary Callus production along with caulogenesis was obtained from leaf explants of micropropagated clonal Eucalyptus grandis after six to twelve weeks of culture. Out of eight clones tested, six were amenable to shoot production using simple media containing naphthaleneacetic acid and either 6-benzyladenine or zeatin. Differences in growth regulator requirements for organogenesis were observed between different clones. These shoots were then elongated on a medium containing gibberellic acid and rooted using media derived from the micropropagation medium. Light conditions were also found to be important for regeneration. This protocol is the first published on regeneration from nonseedling material and it will facilitate the Agrobacterium tumefaciens -mediated transformation of selected clonal Eucalyptus grandis.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - PAR photosynthetically active radiation - PVP polyvinyl pyrrolidone - SH Schenk and Hildebrandt (1972) medium     

The cytokinin-like effect of a lowered temperature on the micromorphology ofNicotiana tabacum L.

The lowering of the cultivation temperature allows one to alter the growth intensity and micromorphology of tobacco cell strains specifically. By a long-term low temperature treatment the effect is deepened, by transferring inocula into normal cultivation temperature it is repaired. Both the growth and morphogenic effects of the low temperature correspond to those of cytokinins, exhibiting even the same strain specificity.     

Effect of N-Methyl-N-nitrosourea and N-Methyl-N-nitrosourethane upon the growth of tissue cultures ofNicotiana tabacum L.

N-Methyl-N-nitrosourea and N-methyl-N-nitrosourethane at concentrations of 0.1 mM to 1 mM inhibited the growth of tissue cultures ofNicotiana tabacum L. The inhibitory effect was proportional to the mutagen concentration applied. The primary expiants (pith slices) and a 3-year tissue culture strain exhibited a different sensitivity to the same mutagen concentrations. The variability in sensitivity of tissue culture inocula to mutagen effects was reduced by previous fractionation of the culture and by standardization of the age and size of inocula. The changes investigated in the ratio of relative growth rates between the controls and treated cultures give evidence of a fluctuating expression of mutagen effect in the course of the subculture interval and may demonstrate a recovery of the cultures from the mutagen effect.     

Regeneration of plants from mesophyll protoplasts ofNicotiana plumbaginifolia Viv.

Summary Protoplasts ofNicotiana plumbaginifolia were isolated by a one step enzymatic method. They were cultured in Ohyama and Nitsch's medium supplemented with 2,4-D (1.0 mg/l), benzylaminopurine (1.0 mg/l) and 14% sucrose. Cell divisions were initiated after 5 days and within 3 weeks colonies were discernible without the microscope. After transfer to Murashige and Skoog's medium containing IAA and kinetin the colonies differentiated into plantlets.     

Somatic embryogenesis from stem and leaf explants of Quercus robur L.

Internodal and leaf segments from pedunculate oak (Quercus robur L.) seedlings were used as explant source to induce somatic embryogenesis. Auxin treatment influenced embryogenic response, which only occurred in explants initially cultured on media containing 4 mg/l naphthaleneacetic acid (NAA) and different benzyladenine (BA) concentrations. After 6 weeks of culture on induction medium, the explants were transferred to medium supplemented with 0.1 mg/l BA and 0.1 mg/l NAA, and 4 weeks later, they were subcultured in a growth-regulator-free medium, in which somatic embryos arose through indirect regeneration on the surface of a nodular callus. Somatic embryos were induced in explants of two out of four seedling provenances. The induction frequency ranged from 16% in leaf explants to 4% in internodal explants. Somatic embryos developed two cotyledons, which were translucent or opaque-white in appearance, but anomalous morphologies were also observed. Different embryogenic lines were established and maintained by repetitive embryogenesis in multiplication medium containing 0.1 mg/l BA plus 0.05 mg/l NAA. These results indicate that tissues from explants other than Q. robur zygotic embryos are able to produce embryogenic cultures. Received: 14 July 1998 / Revision received: 2 November 1998 / Accepted: 6 November 1998     

In vitro morphogenesis from excised leaf explants of Digitalis obscura L.

The morphogenic capacity of Digitalis obscura leaf explants cultured in vitro has been studied, noting factors promoting the differentiation of roots, buds and shoots as well as those promoting callus proliferation. Complete plant regeneration was obtained only by first culturing the leaf explants in a medium with NAA and BA to induce formation of buds, and subsequently transferring them to a medium without growth regulators to achieve the further development of shoots.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - NAA naphthaleneacetic acid     

Regeneration of plantlets from leaf and petiole explants of Pelargonium x hortorum

Summary The in vitro plant regeneration potential of vegetatively propagated geraniums (Pelargonium x hortorum) has been investigated. Using various combinations of growth regulators and a choice of different explants, a regeneration protocol has been developed to raise in vitro plantlets from young petiole and leaf explants from three different cultivars of geraniums. In all three cultivars, very young petiole explants exhibited a higher regeneration potential as compared with leaf explants. Regeneration efficiencies were found to be highly dependent on the cultivar, with cv. Samba showing the highest regeneration potential, followed by cvs. Yours Truly and then Sincerity. Samba also showed the highest number of shoots from both the petiole [57 shoot buds per petiole explant in the presence of 3 μM zeatin and 1 μM indole-3-acetic acid (IAA) and leaf explants (43 shoots per leaf explant with 10 μM zeatin and 2 μM IAA). Shoot buds transferred to Murashige and Skoog (MS) medium supplemented with 0.44 μM N⁶-benzyladenine and 0.11 μM IAA grew vigorously and attained 1–2 cm in length in 3–4 wk. These shoots rooted with 100% efficiency on MS basal medium, and plants developed that showed normal growth and flowering under greenhouse conditions.     

Microtubules and actin filaments co-localize in pollen tubes ofNicotiana tabacum L. andLilium longiflorum Thunb.

Summary Double labeling with fluorescent probes showed that in the cortical cytoplasm of pollen tubes ofNicotiana tabacum andLilium longiflorum, the microtubules and actin filaments co-localize for the most part. They displayed complex net-axial or helical distributions. They structural association of microtubules and actin filaments implies a functional relationship with respect to organelle movement and/or the organization of the cortical cytoplasm and cell surface.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetra-acetic acid - FITC fluoresceine iso-thiocyanate     

Poly(A)+ RNA synthesis in germinating pollen ofNicotiana tabacum L

Poly(A)⁺RNA is synthesized during the first hours of pollen germination and is rapidly incorporated into polysomal structures. After a 2-h pulse with uracil-¹⁴C, 42% of the transcribed fraction of polysomal RNA is polyadenylated. Following 4 h of germination the amount of the newly-made poly(A)⁺RNA decreases steadily at the rate of about 14% per h, whereas that of rapidly-labelled poly(A)⁻RNA continues to grow. Beginning 1 h of cultivation the ratio of poly(A)⁻/poly(A)⁺RNA increases exponentially. Similarly as in non-polyadenylated mRNA the main portion of the synthesized polysomal poly(A)⁺RNA sediments at a rate of 4 to 14 S and its mean size decreases slightly with the time of labelling. RNA isolated from nuclei and cell wall containing pollen tube fraction differed from the polysomal one in higher apeoific radioactivity and the polyadenylated RNA exhibited higher size distribution. The comparison of the results with earlier observations suggests the involvement of poly(A)in mRNA translation in pollen tubes.     

Ultrastructure of embryogenic pollen ofNicotiana tabacum var. Badischer burley

Summary Pollen grains capable of embryogenesis were selectively isolated from (a) near-mature buds from plants induced to flower in short days and low temperature (8 hours light and 18 °C) and (b) young buds from these plants with an additional low temperature treatment (10 °C for 10 days) and fixed for electron microscopy. The pollen from the former formed embryos at a very low frequency in culture, and at the subcellular level showed different degrees of regression of cytoplasm and mitochondria. On the contrary, cold-treated pollen were characterized by a high frequency of embryogenesis, up to 25% of the cultured pollen. They did not show regression of cytoplasm or organelles but had an attenuated cytoplasm which was not rich in ribosomes. Another noteworthy feature of embryogenic grains was the condensed nature of mitochondria. These characteristics of embryogenic grains indicate that they are repressed for gametophytic differentiation. The embryogenic pollen did not differentiate from gametophytic pollen which were very distinctive, having a thick exine, and dense cytoplasm rich in ribosomes. The close similarity of embryogenic grains with young microspores in terms of thin exine and sparse cytoplasm is suggestive of an indeterminate state and that determination into gametophytic or sporophytic (embryogenic) type is probably the function of differential gene activity. Of interest, in this context, is the condensation of mitochondria in embryogenic grains. The relationship, if any, between mitochondrial condensation and embryogenesis remains to be resolved.     

Immunoelectron microscopy of myosin associated with the generative cells in pollen tubes ofNicotiana tabacum L.

In this study, polyclonal anti-myosin antibodies were used for immunogold labeling of ultrathin sections of pollen tubes ofNicotiana tabacum L. to unravel the ultrastructural localization of myosin associated with the generative cells. Clusters of immunogold particles were consistently found in association with the area of the outer surface of the vegetative cell plasma membrane present around the generative cell. Compared to the generative cell cytoplasm, the nucleoplasm showed higher numbers of gold particles. This is the first direct evidence demonstrating the presence of myosin in the nuclei of the generative cell of flowering plants. The possible implications of these findings are discussed in relation to movement of the generative cell in the pollen tube cytoplasm.     

Regeneration of plantlets from in vitro raised leaf explants of Cleisostoma racimeferum Lindl

Protocorm like bodies (PLBs), callus and shoot buds developed in culture from in vitro raised foliar explants of Cleisostoma racimeferum. Among the different basal media, better result was obtained on MS medium containing sucrose (3%) and BA (2 microM) with approximately 80% frequency after 40 days of culture. Young leaves (15 week old) produced better PLBs. Whole leaf placed vertically upside-up orientation can regenerate PLBs and shoot buds (80%). PLBs and shoot buds formed on entire surface of the leaves. Cultures on BA and NAA (2 and 2 microM respectively in combination) stimulated callus mediated regeneration (68%). The rooted plantlets regenerated within 8-10 week from PLBs and shoot buds on MS medium containing IAA and kinetin (2 microM each in combination). BA containing medium triggered multiple shoot bud formation, while NAA alone or in combination with other growth regulators was inhibitory. Incorporation of activated charcoal (0.01%) in the medium stimulated formation of repetitive PLBs and multiple shoot buds. Rooted plants were ready for harvest after 20-22 week of initiation of culture. About 65% of the potted plants survived after 3 months in the poly house.     

Some factors affecting the seed set afterin vitro pollination of excised placentae ofNicotiana tabacum L.

The paper deals with the effect of some factors on the set of mature seeds in artificially pollinated excised placentae cultivatedin vitro. It was revealed that both the degree and the variability of the seed set depend on the age of the experimental flowers. The best results were achieved using placentae excised from the flowers collected 1 to 3 days after anthesis. The technique of pollination which consists in applying pollen onto a certain site of the placenta was substantially more successful than dusting pollen over the entire placenta surface or sowing pollen on the nutrient medium in close proximity to placentae. It was confirmed that casein hydrolysate at a concentration of 500 μg ml^?1 nutrient medium favourably affected the seed formationin vitro. The seed set resulting fromin vitro fertilization does not depend upon whether, during, a fourweek cultivation, the cultures are kept in the dark or in diffused daylight.