O. L. Gamborg

Plant protoplast fusion and growth of intergeneric hybrid cells

SummaryInterspecific and intergeneric fusions of plant protoplasts were induced by polyethylene glycol (PEG) 1540 or 4000. The frequency of heterokaryocyte formation (or rate of fusion) was much higher when PEG was eluted with a high pH-high Ca2+ solution or a salt solution than when it was eluted with a protoplast culture medium. The frequency of heterokaryocyte formation was also affected by the types of enzymes used for wall degradation, duration of enzyme incubation and molality of the PEG solutions.The maximum frequency of heterokaryocyte formation was 23% for V. hajastana Grossh.-soybean (Glycine max L.) and barley (Hordeum vulgare L.)-soybean, 35% for pea (Pisum sativum L.)-soybean, 20% for pea-V. hajastana, 14% for corn (Zea mays L.)-soybean and 10% for V. villosa Roth-V. hajastana.40% of the barley-soybean, corn-soybean and pea-soybean heterokaryocytes divided at least once. Some divided many times and formed clusters of up to 100 cells in 2 weeks. The heterokaryocytes of soybean-V. hajastana, V. villosa-V. hajastana also divided. Of the PEG-treated protoplasts of N. langsdorffii and N. glauca 13.5% developed into tumor-like calli. The morphology of these calli was very much like that of the tumors produced on amphidiploid plants of N. langsdorffii x glauca.Nuclear staining indicated that heterokaryocytes of V. hajastana-soybean, pea-soybean, corn-soybean and barley-soybean could undergo mitosis. Nuclear divisions in a heterokaryocyte were usually synchronized or almost synchronized. Nuclear fusion and true hybrid formation usually occurred during the first mitotic division after protoplast fusion. A hybrid of barley-soybean in third cell division was observed. The frequency of heterokaryocytes which underwent nuclear fusion has not been determined. Multipole formation and chimeral cell colonies were also observed.

Callus formation and plant regeneration from mesophyll protoplasts of rape plants (Brassica napus L. cv. Zephyr)

Abstract Protoplasts from mesophyll cells of rape plants ( Brassica napus L. cv. Zephyr) were isolated by enzymatic removal of cell walls in an osmoticum consisting of sorbitol and mannitol. A two-step enzyme treatment involving 0.5% each of Onozuka P1500 cellulase and Rhozyme HP150 hemicellulase followed by 0.5% each of Driselase and hemicellulase at pH 6.2 resulted in the production of viable protoplasts in 80–90% yield. The protoplasts were cultured in droplets of B5 medium in petri dishes at a light intensity of 100 lux and 26°. They were able to regenerate cell walls. Cell division was apparent after 30 h of culturing and by 60 h up to 38% of the protoplasts had divided once. Cell clusters were formed within 15 days of culturing. The calli originating from cell clusters were induced to differentiate and to form complete plants.

Cell division and plant development from protoplasts of carrot cell suspension cultures.

SummaryCell regeneration and sustained division have been observed in protoplasts from carrot cell suspension cultures. Carrot plants were produced from the protoplasts by embryogenesis.

Morphogenesis and plant regeneration from callus of immature embryos of sorghum

Abstract Callus with leafy shoots were obtained from immature embryos of sorghum (Sorghum bicolor L. Moench) collected 12–18 days after pollination. The embryos were cultured on a defined agar medium containing Murashige-Skoog (MS) mineral salts supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 10–50 μM zeatin. The callus was produced within 10 weeks and further proliferated after subculture on the same medium. Small sections of callus with leafy shoots were transferred onto the same nutrient medium but containing 1–1 μm indoleacetic acid (IAA). Plantlets produced after 8–10 weeks were transferred to pots and grown to maturity. Some plants were sterile while others produced fertile seeds. Meiotic metaphase examination of 25 panicles revealed chromosome number of 2n = 20 corresponding to normal plants with 10-10 anaphase distribution.

The effect of hormones on anthocyanin accumulation in cell cultures of Haplopappus gracilis.

SummarySuspension cultures of Haplopappus gracilis accumulated anthocyanin when grown in defined media with 4.5×10-6M 2,4-D. Transfer of cells to media with 10-5M kinetin or benzyladenine and no auxin or 10-7M NAA for 6 days resulted in increased anthocyanin concentration of the cells but the total amount of pigment was unaffected due to differences in growth rates. The cultures yielded up to 35 mg pigment per gram dry weight.Cells grown in batch culture in media with 10-5M kinetin and with 10-7 M NAA or 5×10-5M NAA sampled and analyzed daily grew at the same rate. The concentration of anthocyanin differed, being lower in cells at 5×10-5M NAA. After 6 days there was a rapid increase in pigment formation, and by 14 days the concentration of anthocyanin in cells in the two media were the same.When the cells were cultured in 3.5-1 phytostats and 600 ml culture was replaced daily with 600 ml medium, anthocyanins accumulated when the NAA concentration was 10-7M but not at 10-6M. At 10-7M NAA the cultures remained pigmented and anthocyanin accumulation could be restored after a temporary loss of pigmentation due to an earlier, higher auxin concentration. The changes in concentration of phenylalanine ammonia-lyase did not correspond to changes in the rate of anthocyanin accumulation. The enzyme showed a maximum 4–8 h after inoculation of cells to fresh media. Cells grown on agar plates and rich in anthocyanin were observed to divide without loss of pigmentation, demonstrating that cells differentiated with respect to anthocyanin production undergo mitosis.

Regeneration of cassava plants from apical meristems

Abstract Cassava plants were regenerated from meristems of five cultivars; Colombia No. 800, Llanera, Venezuela No. 255, Ecuador No. 133 and Mexico No. 35. Using benzyladenine (BA), gibberellic acid (GA3) and naphthaleneacetic acid (NAA) at molar concentrations of 5·.10−7, 10−7 and 10−7, Respectively, resulted in complete plant development on Murashige-Skoog (MS) medium supplemented with vitamins as in B5. GA3 in combination with NAA resulted in root formation, while BA with NAA produced callus and storage roots. The meristems were cultured in a growth cabinet at 26°, 60% relative humidity, and exposed to a light intensity of 4000 lux from cool, white fluorescent lamps using a light and dark cycle of 18/6 h.

Freeze-preservation of pea meristems in liquid nitrogen and subsequent plant regeneration

Abstract A procedure has been developed for the freeze-preservation of pea ( Pisum sativum L.) meristems and subsequent plant regeneration. The meristems were precultured in the presence of 5% DMSO for 48 h followed by freezing at cooling velocities varying from 0.5 to 1.0°C/min up to a terminal freezing temperature of −40°C and subsequently stored in liquid nitrogen (−196°C). Maximum survival as determined by the regrowth of meristems into whole plants was obtained at a cooling rate of 0.6°C/min after 1 h storage in liquid nitrogen (73%). The meristems were in storage in liquid nitrogen for up to 26 weeks and upon reculture more than 60% of the meristems differentiated into whole plants. Cold conditioning of meristems at 4°C prior to freezing without preculture in the presence of DMSO resulted in lower survival rates. Glycerol and ethylene glycol were ineffective cryoprotectants. Ultra-rapid freezing of meristems by direct immersion in liquid nitrogen was also lethal.

Morphogenetic Investigations on in vitro Leaf Culture of Tomato (Lycopersicon esculentum Mill. cv. Starfire) and high Frequency Plant Regeneration

Summary Leaf explants of tomato ( Lycopersicon esculentum Mill . cv. Starfire ) were cultured in vitro on Murashige and Skoog medium under controlled environmental conditions. Growth and morphogenetic responses of the explants to cytokinins and auxins were compared at different concentrations and combinations. Combinations of benzyladenine (BA) and indoleacetic acid (IAA) were more effective in inducing shoot differentiation than those of BA and naphthaleneacetic acid (NAA). Multiple shoot differentiation on the calli occurred at 10 μM BA with various concentrations of IAA. Rooting of the differentiated shoots was readily achieved by culturing the shoots on medium containing no growth hormones. Kinetin (K) in conjunction with IAA or NAA failed to induce shoot differentiation. Shoot and root differentiation also occurred at 0.1 μM zeatin (Z) + 10 μM IAA or 1 μM Z + 0.1 μM IAA. When used alone, BA or Z at 5 μM induced complete plant regeneration from the callus. Large numbers of regenerated plantlets were transferred to pots and grown to mature plants.

Bean mesophyll protoplasts: production, culture and callus formation☆

Abstract High yields of viable protoplasts were produced from leaf tissue of Phaseolus vulgaris L. var. Pinto. Consistent production of protoplasts depended on enzymatic liberation of the protoplasts near pH 7.0. Cultured protoplasts regenerated cell walls within 24–48 h after isolation. Cell division was observed within 72 h after isolation and sustained division led to callus formation.

Regeneration of the liverwort Marchantia polymorpha L. From protoplasts isolated from cell suspension culture

Abstract A cell suspension culture was initiated from green, haploid callus of Marchantia polymorpha L. Protoplasts were isolated from cultured cells by enzymatic degradation of cell walls. The first division of the cells regenerated from protoplasts was observed after 7–10 day culture. Callus and regenerated plants (thalli) from protoplasts were obtained by gradually reducing the osmolarity in the medium and subsequently culturing regenerated cells on an inorganic phytohormone-free medium.