F. Constabel

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.

Stimulation of Sanguinarine Accumulation in Papaver somniferum Cell Cultures by Fungal Elicitors

Abstract Cells of a seven year old strain of Papaver somniferum L. when cultured for 2 weeks and incubated with substances known to elicit the formation of phytoalexins, responded by turning reddish brown within 6 h and accumulating sanguinarine. Morphinan alkaloids were not detected. Media (100 ml) containing 1 ml of Botrytis spec. preparations raised the level of sanguinarine in the cells 26 times over the maximum level found in controls. Over a culture period of 79 h the cells achieved a sanguinarine concentration of 2.9% of dry weight. Media (100 ml) with 1 ml of Rhodotorula rubra preparation, 15 mg arachidonic acid, 1 mg actinomycin, 0.5 ml of Helminthosporium gramineum, Sclerotinia sclerotiorum , or 5 ml Colletotrichum gloeosporoides preparation elicited a considerable, but relatively weaker response. Sanguinarine accumulation was also found to occur in the medium and reached a concentration of 43% of total sanguinarine per culture when cells were cultured in 100 ml medium with 5 ml Colletotrichum preparation for 24 h. Young poppy cell cultures initiated 9 months ago responded to the presence of Botrytis material as did 7-year-old cultures.

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.

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.

Elicitor-stimulation of monoterpene indole alkaloid formation in suspension cultures of Catharanthus roseus

Abstract Upon treatment of 5 cell lines of Catharanthus roseus with homogenates of various fungi, as well as with chemically defined phytoalexin elicitors, all except one (non-alkaloid producing #916) responded with browing and accumulation of tryptamine within 6 – 24 h. Cells of line #615 responded with not only accumulating tryptamine, but also N-acetyl tryptamine, strictosidine lactam, ajmalicine, tabersonine, lochnericine, and catharanthine. Based on amounts of alkaloids accumulated, cells of line #615 performed best when treated with homogenates of Alternaria zinnae, Pythium apbanidermatum, Verticillium dabliae, and Rhodotorula rubs. A Pythium homogenate concentration of 5 % and a Rhodotorula homogenate concentration of 0.5 % effected maximum alkaloid yields, and, thus, were used in subsequent studies. These revealed a temporary increase of the level of alkaloids in cells and in their medium after 12 – 24 h of treatment. Ten-day-old subcultures responded better than younger and older ones. The elicitor stimulated accumulation of alkaloids and alkaloid composition did not depend on the use of 1-B5 or alkaloid production medium. A 5 l cell suspension of #615 grown in a 7.5 l bioreactor and treated with 5 % Pythium homogenate for 18 h was found to contain strictosidine lactam, ajmalicine, and catharanthine in concentrations of 27, 10, and 13 μg/g DW respectively, the medium contained 42 % of total ajmalicine.

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.

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.

Regeneration of pea (Pisum sativum L.) plants from shoot apical meristems

Summary A procedure has been developed to obtain complete plants from meristems of three cultivars of Pisum sativum L., namely «Century», «Laxtons Progress» and «Afghanistan». Benzyladenine (BA) alone or in combination with naphthaleneacetic acid (NAA) at molar concentrations of 5 × 10 −7 and 10 −6 , respectively, induced shoot differentiation in meristems cultured on B5 medium at 26° C, 60 % relative humidity and exposed to fluorescent light (4000 lux, photoperiod 18 hrs). When applied alone, NAA induced complete plant formation. Root formation, on the shoots produced by culturing meristems, was induced by reculturing the shoots, 2 cm long, on half strength B5 medium supplemented with NAA at a concentration of 10 −6 M.

Alkaloid production in Catharanthus roseus cell cultures : XII. Biosynthetic capacity of callus from original explants and regenerated shoots.

Callus derived from hypocotyls of periwinkle, Catharanthus roseus, responded to culture on nutrient media supplementedwith IAA, BA, and zeatin with shoot formation at low frequencies. However, shoot regenerating callus could be very successfully propagated and subcultured. Alkaloid profiles of callus derived from the original explants (hypocotyls) as well as callus derived from regenerated shoots were almost identical. Subcultures of old callus (initiated in 1978) failed completely to grow shoots. In programs for long-term preservation of alkaloid producing cell lines by regeneration and storage of shoots, selection for ability to form shoots would have to precede selection for alkaloid production.