Tage Eriksson

The development of somatic embryos in tissue-cultures initiated from immature embryos of Picea abies (Norway spruce)

Embryos of Picea abies at various developmental stages were cultured on defined media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (10−5 M) and N6-benzyladenine (BA) (5×10−6 M). The immature embryos gave rise to a highly friable and embryogenic callus which could be maintained by subculture and contained polarized and organized structures (somatic embryos) consisting of long highly vacuolated cells at one end (suspensor) and a group of small meristematic cells at the other (embryonal end). These structures closely resembled the early stages of normal zygotic embryogeny. Upon further culture these structures formed a bipolar shoot-root axis with an independent and closed vascular system. In many instances either the shoot or the root meristems failed to differentiate. Embryogenic tissues obtained on agar media could be transferred to liquid media and maintained by subculture for at least 6 months. The development of somatic embryos was observed in the liquid cultures also.

Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium tumefaciens — mediated gene transfer

SummaryA transformation system that allows regeneration of transgenic pea plants from calli selected for antibiotic resistance was developed. Explants from axenic shoot cultures and seedling epicotyls were cocultivated with nononcogenic Agrobacterium tumefaciens strains, and transformed callus could be selected on callus-inducing media containing either 15 mg/l hygromycin or 75 mg/l kanamycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on hygromycin-resistant calli, but not on the calli selected for kanamycin resistance. Regenerated shoots could subsequently be rooted and transferred into the greenhouse. In addition, the effects of different callus-inducing and growth media on organogenesis were investigated. The transformation of the calli and regenerated plants was confirmed by DNA analysis.

The induction of aggregation and fusion of Dancus carota protoplasts by polyethylene glycol

Summary A method for induction of high aggregation and fusion frequency is described. About 10% dinucleated fused protoplast were obtained if Daucus carota protoplasts from a normally dividing cell suspension in protoplast growth medium were treated in a final concentration of 28% (w/w) polyethylene glycol at 25°C. The influence of salt composition and concentration on protoplast aggregation and fusion was studied as well as effects of different temperatures, pH and protoplast age and condition. Light- and electron micrographs show the strong adhesion and fusion of cell membranes and the osmotic changes in the protoplasts induced by polytehylene glycol.

Effect of agar concentration on growth and anatomy of adventitious shoots of Picea abies (L.) Karst

The development of adventitious shoots of Picea abies was affected by the agar concentration in the culture medium. Increasing the agar concentration from 0.5 to 2.0% decreased vitrification, but at the same time reduced shoot growth and rooting potential. Slightly vitrified plantlets could become acclimatized to greenhouse conditions. The mesophyll of needles developed in vitro was interspersed with large air spaces; the lower the agar concentration, the larger the air spaces. After transfer to the greenhouse, the new needles from the acclimatized plantlets had an anatomy approaching that of plants growing in field.

Transfer of algal chloroplasts into protoplasts of higher plants

SummaryPolyethylene-glycol treatment of mixed suspensions of isolated protoplasts and of chloroplasts induces chloroplast uptake by the protoplasts. Chloroplasts of algal origin (Vaucheria dichotoma (L.) Ag.) were transferred with high frequency into protoplasts of Daucus carota L.

Nuclear division in isolated protoplasts from cells of higher plants grown in vitro

SummaryProtoplasts were isolated from cell suspensions of Haplopappus gracilis. The cell walls were degraded by the cellulase preparation “Onozuka P 1500” at a concentration of 5%. Sorbitol was found to work well as osmotic stabilizer in concentrations of 0.4–0.6 M. The protoplasts were cultured in growth medium after isolation; 3–5% went through nuclear division once and less than 1% also for a second time. No nuclear fusion was observed.

Improved Culture Ability of Potato Protoplasts by Use of Activated Charcoal

Protoplasts were obtained from in vitro grown plants of Solanum tuberosum L. The protoplasts were cultured in X-plate petri dishes with the culture medium joined to a reservoirmedium. When activated charcoal was added to the reservoirmedium the culture ability of the protoplasts was significantly increased. The effect of activated charcoal was mainly due to a less pronounced browning of the developing protoplasts and this technique might be of help in protoplast cultures where browning is a problem.

Enucleation of Plant Protoplasts by Cytochalasin B

Summary On exposure of plant protoplasts to cytochalasin B under high speed centrifugation, the nucleus, surrounded by some cytoplasm and a plasma membrane, is pinched off. Subprotoplasts containing nuclei are formed, together with healthy enucleated protoplasts. By varying the concentration of cytochalasin B and centrifugation speed, enucleated protoplasts could be prepared from Nicotiana sylvestris, Nicotiana tabacum, Pisum sativum and Daucus carota . Nucleated subprotoplasts divided at high frequency in a suitable medium.

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants

SummaryAn analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.

Nuclear DNA-content during the initiation of callus formation from isolated protoplasts of Solanum tuberosum L

Abstract Cytofluorimetric measurements of the DNA-content in the nuclei during the initiation of callus formation from isolated protoplasts of potato were performed using propidium iodide as fluorochrome. Reproducible measurements were obtained after digestion of the cell walls and after RNase treatment. Nuclei with high DNA-content were found early in the culture and the frequency of these nuclei increased with time in culture. In addition to highly replicated nuclei, multinucleated cells were found, the frequency of which decreased during prolonged culturing.