In vitro propagation of Amaryllis belladonna

Amaryllis belladonna L. plants were multiplied successfully by means of tissue culture techniques. Different plant parts were tested as explant material, but plantlets could only be generated from the twin-scales and immature scapes. These in vitro-formed plantlets were divided into four parts and used for further multiplication. The twin-scale explants had the highest multiplication rate when a medium with 22.2 M benzyladenine and 0.54 M naphthaleneacetic acid was used. The sucrose concentration played an important role in the initiation of new plantlets, and the best results were obtained when a sucrose concentration of 2–3% was used. Anatomical observations were made during the initiation of the new plantlets.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - Benomyl (methyl [1-[(butylamino) carbonyl]-1H-benzimidazol-2-yl] carbamate) - Folpet (2-[(trichloromethyl)thio]-H-isoindole-1,3(2H)-dione phthalimide(I))     

Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.)

Immature and mature nonstratified seeds of white ash (Fraxinus americana L.) were dissected transversely and 2/3 of each seed was placed onto agar-solidified Murashige and Skoog medium. Adventitious buds, shoots, and somatic embryos formed on callus, cotyledons, and hypocotyls of the resulting seedlings. Shoot organogenesis was induced on explants cultured on medium with 10 M thidiazuron but not on explants on media with benzyladenine (BA) or isopentenyladenine. Not all seed sources were equally capable of shoot organogenesis and embryogenesis. Atypical of adventitious regeneration of other woody plants, mature seed explants of white ash were more organogenic with shoots that elongated better than explants from immature seeds. Somatic embryogenesis was observed in cultures where mature seeds were first cultured for 4 weeks on a medium containing 10 M adenine 2,4-dichlorophenoxyacetic acid in combination with 0.1 and 1.0 M thidiazuron, followed by transfer to a medium containing 0.05 M 6-benzyladenine and 0.5 M naphthaleneacetic acid. Adventitious shoots and epicotyls from both seedlings and germinated somatic embryos were rooted under intermittent mist and acclimatized to the greenhouse.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - TDZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-ylurea - WPM woody plant medium     

Microspore cultures as donor tissue for the initiation of embryogenic cell suspensions in barley

We have initiated embryogenic cell suspension cultures of barley (Hordeum vulgare L.) Igri from isolated microspore cultures. Data were obtained on the time required for establishment, frequency of establishment, i.e. number of calluses out of the total number of initiations giving rise to suspensions, and embryogenic capacity of the suspension cultures. For comparison, establishment of embryogenic cell suspensions from callus derived from immature zygotic embryos of Igri, Dissa and Golden Promise was also carried out. The results revealed that embryogenic suspension cultures were established in half the time and with a seven-fold higher frequency from microspore cultures than from zygotic embryo-derived calluses. The suspension cultures were still capable of embryo formation after two years. However, only albino plantlets were regenerated. For comparison, long term callus cultures derived from microspores, anthers and zygotic embryos were established. From the anther and zygotic embryo-derived callus cultures green plants were continuously regenerated, whereas the microspore-derived callus cultures lost this ability after the second subculture.     

Photoautotrophic growth of soybean cells in suspension culture. II. Optimization of culture medium and conditions

We have attempted to optimize the conditions under which a photoautotrophic soybean suspension culture line (SB-P; Horn et al. 1983) is grown. Magnesium, phosphate, and calcium concentrations were varied individually from one-tenth to five times the normal level found in the Murashige and Skoog (1962) recipe. After two subcultures, only phosphate at one-tenth the normal level caused the cells to show a substantial reduction in fresh and dry weight increase and chlorophyll level. Nitrate and ammonium levels were inversely varied in 20 millimolar increments of potassium nitrate and ammonium chloride. Neither N-source alone could support growth through two subcultures. A ratio of 40 millimolar potassium nitrate to 20 millimolar ammonium gave significantly better fresh and dry weight increases than did a ratio of 20:40 or 30:30 but the chlorophyll level was unchanged. The minor salts as a group resulted in a small improvement in growth when provided at twice the normal level.Indole-3-acetic acid at five milligrams per liter resulted in significantly better fresh and dry weight increases than did -naphthaleneacetic acid at any level but the final chlorophyll level was not changed. There was no correlation between growth and kinetin level and this resulted in the discovery that SB-P cells are cytokinin-autotrophic, as are heterotrophic SB cells, with regard to both growth and greening ability. Growing SB-P cells under a 14 h:10 h day:night photoperiod resulted in a slow but inevitable death. Increasing the carbon dioxide level to 10% for four weeks gave no increase in SB-P cell growth or chlorophyll level, but SB-P cells would not grow with carbon dioxide levels below 0.4%. The results clearly show that SB-P cells, despite their tenuous existence, are capable of adapting to a wide range of culture conditions. A simplified and improved culture medium for photoautotrophic cultures is given.Abbreviations SB-P photoautotrophic soybean cells - SB-M photomixotrophic soybean cells - SB-H heterotrophic soybean cells     

Tissue culture and plant propagation of Gomphrena officinalis — a Brazilian medicinal plant

Leaf and stem segments of Gomphrena officinalis originated from aseptically grown seedlings were used to initiate cultures. Callus production was obtained on gelled Murashige & Skoog medium supplemented with 6-benzylaminopurine alone (1.0, 5.0 or 10.0 mgl^-1) or combined with -naphthalene acetic acid (0.1, 0.5 and 1.0 mgl^-1) after 10 to 15 days of culture, and can be transferred to fresh medium every 30 days. The combinations of 5.0 or 10.0 mgl^-1 of 6-benzylaminopurine with 0.1 mgl^-1 of -naphthalene acetic acid were found to be the best for shoot regeneration. Adventitious shoot formation occurred after 50 to 60 days of culture in leaf and internode stem explants. Nodal segments developed actively growing lateral buds after 30 days of culture. Gelled Murashige & Skoog medium containing 10 mgl^-1 of indole-3-butyric acid was considered optimal for the rooting of shoots. Rooted plants transferred to potting soil could be successfully established.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog - NAA -naphthalene acetic acid     

Assessment of inter- and intra-population variation in quinoline alkaloid profile of juvenile shoot cultures of Cinchona ledgeriana: effect of method of nutrient delivery on alkaloid profile

Intra-population quinoline alkaloid profiles surveying quinine, quinidine, cinchonine and cinchonidine were determined for each of five populations of Cinchona ledgeriana grown as shoot-culture for 125 days. No significant difference in respect of mean alkaloid content between populations was detected. In contrast, there was considerable between-seedling variation in alkaloid content within each population. When nutrients were delivered to shoot-cultures in droplet form by means of an aerosol spray (as compared to the supply of nutrients direct from agar-or liquid-based reservoirs) alkaloid profile was greatly perturbed; most notable in this respect was a four-fold increase in the production of cinchonidine concomitant with a four-fold decrease in the production of cinchonine. These data are discussed with reference to the optimisation of quinoline alkaloid production by juvenile shoot-cultures of Cinchona ledgeriana.     

Functional properties of ficoll and their influence on anther culture responses of wheat

The effects of ficoll in liquid culture media have been contradictory in previous reports. The objective of this study was to determine the functional properties of ficoll in potato 4 (P4) liquid induction medium and their influence on anther culture responses of wheat. Ficoll addition significantly (p0.01) reduced callus production from the anthers of spring wheat cv. Pavon 76. The reduction was directly related to the concentration of ficoll added within the range of 50 to 200 g l^-1 medium. Although the addition of ficoll significantly (p0.01) increased the percentage of regenerable calli and the ratio of green vs. albino plants, the final yield of green plants per 100 anthers was significantly lower. Consistent results also were obtained with four other spring wheat genotypes (Chris, Butte 86, WA 6916, and Edwall). Ficoll concentration affected the density, viscosity, and osmolality of the liquid media. The higher medium density caused by ficoll addition increased the percentage of floating calli, as well as the percentage of regenerable calli and the ratio of green vs. albino plants. However, the increased medium viscosity by ficoll addition significantly (p0.01) reduced callus production. Ficoll addition also increased medium osmolality, which affected callus production by interacting with the sugar concentration of the induction media. Using response functions, the estimated maltose concentration for maximum callus production was 105 g l^-1 for the standard P4 media, compared with 68 g l^-1 for the ficoll-containing P4 media. These results clearly demonstrate that ficoll addition to the liquid P4 induction medium containing high sucrose concentration (90 g l^-1) is deleterious to the maximum production of green plants from wheat anther culture.     

Anther culture responsiveness of Hordeum spontaneum derived spring barley lines and a genetic analysis of plant regeneration

Anther culture responsiveness of three H. spontaneum derived spring barley lines, RS170-47(A), RS20-1(B) and 1B-152B(C) was investigated using only one type of culture medium and treatment. The line 1B-152B was identified as highly responsive producing 22.4 total and 12.4 green regenerants per 100 anthers plated. 74% of these green regenerants were spontaneous double haploids. A genetic analysis involving F1 and F2 plants derived from crosses A × B and B × C revealed that the factor(s) determining high anther culture responsiveness in line 1B-152B was heritable and behaved as dominant in the F1. There was an indication that genotypic responsiveness in anther culture for green plant regeneration was different from total or albino plant regeneration.     

A study of factors affecting anther culture of cauliflower (Brassica oleracea var. botrytis)

Experiments on three autumn-heading cauliflower genotypes (2 hybrids and a genotype selected from a population) were conducted to study different factors affecting anther culture. Culture conditions of the donor plants proved to be important: the best results were obtained during spring in a greenhouse where the temperature was maintained between 10 and 20°C. Overall winter and spring seemed more suitable than summer and early autumn for culture establishment. The optimal bud development stage depended on the genotype: for the hybrid 702, the greatest number of embryos for 100 plated anthers was obtained at the uninucleate pollen stage of the microspores; for V23.2 and 703, the optimal stage of the buds corresponded to the first mitotic division. Sucrose proved to be the best carbon supply for embryogenesis with an optimal concentration of 140 g l^-1. The addition of a cytokinin (BAP) in the medium led to lower embryo production, and this negative effect increased when the hormone concentration in the medium increased. The use of liquid medium and a dark incubation period immediately after the high temperature treatment were favourable for embryogenesis.     

In vitro evaluation of sugarcane resistance to gumming disease and of Xanthomonas campestris pv. vasculorum aggressiveness

Sugarcane plantlets were sectioned halfway between the base and the youngest ligule and then inoculated by soaking the wound in a suspension of Xanthomonas campestris pv. vasculorum. The infection caused rapid necrosis of the inoculated leaves, chlorosis of uninoculated leaves, or death of the inoculated plantlet. New tillers sometimes showed chlorosis or white streaks. The effects of the inoculum concentration, the cultivar, and the bacterial strain on symptom severity were determined. The ranking of cultivars depended on the inoculum concentration, and strains were found to differ with regard to aggressiveness. However, cultivars and strains were more effectively classified in greenhouse trials. The poor expression of leaf resistance appeared to limit the use of the in vitro test.