Shoot-forming Cultures Research Articles

Cytokinin-induced abnormal shoot organogenesis is associated with elevated Knotted1-type homeobox gene expression in tobacco

The molecular mechanisms that regulate the transcription of key developmental genes involved in shoot organogenesis have yet to be fully elucidated. However, it is clear that plant growth regulators, such as cytokinin, play a critical role in the differentiation of adventitious shoots. In Nicotiana tabacum zz100 leaf discs, high frequency shoot formation could be induced with 5 microM of the cytokinin N6-benzyladenine (BA). Increasing the exogenous BA concentration to greater than 20 microM resulted in stunted explants with abnormal shoot morphology and altered mineral composition. Explants with abnormal shoots did not appear to be hyperhydric. Abnormalities were, however, associated with an increase in the expression of a knotted1-type homeobox gene (TobH1) isolated from normal shoot-forming cultures. The results suggest that the development of cytokinin-induced abnormal shoot morphology possibly involves changes in TobH1 gene expression.

PkMADS1 is a novel MADS box gene regulating adventitious shoot induction and vegetative shoot development in Paulownia kawakamii

Direct regeneration of shoot buds in vitro is an important technique in plant genetic manipulation. We describe the isolation and functional characterization of a novel MADS box cDNA (PkMADS1) from Paulownia kawakamii leaf explants undergoing adventitious shoot regeneration. mRNA gel blot analysis confirmed the expression of PkMADS1 in the shoot-forming cultures, but no signal was observed in the callus-forming cultures. PkMADS1 transcripts were also detected in shoot apices, but not in root apices, initial leaf explants or the flower. In situ hybridization revealed that its expression was restricted to developing shoot primordia in the excised leaf cultures, suggesting a role for this gene in adventitious shoot formation. Transgenic Paulownia plants over-expressing the PkMADS1 gene showed some changes in phenotype, such as axillary shoot formation. In the antisense transformants, shoots were stunted and had altered phyllotaxy, and, in some lines, the shoot apical meristem appeared to have been used up early during shoot development. Leaf explants from the antisense transgenic plants showed a tenfold decrease in shoot regeneration compared with explants from sense transformants or wild-type. Our results show that PkMADS1 is a regulator of shoot morphogenesis.

A differentially expressed bZIP gene is associated with adventitious shoot regeneration in leaf cultures of Paulownia kawakamii

Plant regeneration in tissue culture is important for the successful application of transgenic technology in crop improvement. We isolated a bZIP motif containing cDNA (PKSF1) from excised leaf cultures of the fast-growing tree Paulownia kawakamii by a novel subtractive hybridization strategy. PKSF1 codes for a leucine zipper transcription factor and is preferentially expressed during adventitious shoot regeneration. It showed about a sixfold higher level of expression in the shoot-forming tissues compared to the callus-forming cultures of Paulownia. In situ hybridization analysis showed that PKSF1 expression was localized in the developing shoot primordia of the shoot-forming cultures. It was also expressed in the apical meristem and provascular tissues in the vegetative shoot apex. Quantitative polymerase chain reaction results showed that only basal levels of transcripts were present in callus-forming tissues at day 0 and day 10, whereas maximal expression was seen in shoot-forming tissues at day 10, suggesting that PKSF1 is associated with adventitious shoot bud development in Paulownia.

Inhibition of shoot induction by 5-azacytidine and 5-aza-2'-deoxycytidine in Petunia involves DNA hypomethylation.

Shoot bud regeneration from Petunia leaf disks was inhibited when they were cultured with the demethylating agents, 5-azacytidine (AzaC) and 5-aza-2'-deoxycytidine (AzadC), in shoot induction (SI) medium. Explants induced shoot primordia if they were transferred after 1 week from the medium containing the drugs to medium without drugs. The fresh weight of leaf disks cultured on SI medium for 2 weeks in the presence of the drugs was 60-80% lower when compared to control shoot-forming cultures. Internode length was reduced when shoots were transferred to phytohormone-free Murashige and Skoog medium containing the drugs. However, no other morphological abnormalities were seen in these shoots, even at 20 µM AzaC or 5 µM AzadC. Coupled restriction enzyme digestion (with HpaII and MspI) and random amplification of genomic DNA was performed to detect the level of methylation of CCGG sites in the DNA of the explants exposed to AzaC and AzadC. Over 15 amplified bands were detectable in the control. Five of these bands were absent in the amplified products when digested DNA from the drug-treated explants was used as the template, showing that hypomethylation of DNA had occurred. This suggests that inhibition of shoot bud formation in the presence of the drugs AzaC and AzadC may be due to the altered methylation status.

Nitrogen metabolism in cultured cotyledon explants of Pinus radiata during de novo organogenesis

Nitrogen metabolism was investigated under shoot‐forming (SF) and non‐shoot‐forming (NSF) conditions in cultured cotyledon explants of Pinus radiata by following the incorporation of [14C]‐l,2‐acetate into various metabolites. Early in culture, the lipid fraction contained the most 14C; however, this percentage decreased in favor of increased label in the amphoteric fraction. Label in the amphoteric fraction of SF cultures decreased by day 21 but plateaued in NSF cultures at this time. Radioactive labeling of the principle nitrogen metabolites, glutamate and glutamine, which made up the majority of the amphoteric fraction, paralleled labeling patterns in the amphoteric fraction. Percentage label in glutamate remained at similar levels throughout the 21‐day culture period for both SF and NSF cultures. Specific activity of glutamate (kBq mg‐1) was significantly greater during promeristemoid formation in SF compared to that in NSF tissues. Glutamine labeling increased during shoot bud initiation in SF cultures, but dropped to lower levels during shoot bud development. In contrast, in NSF cultures, there was a continual and substantial increase in glutamine labeling throughout the 21‐day culture period. These trends were similar when the specific activities of glutamine were determined, as there was a continual decrease from culture initiation to the end of shoot bud differentiation in SF cultures. In NSF cultures, in contrast, specific activity of glutamine increased substantially from day 5 to 21 relative to that in SF cultures. The nitrogen assimilation enzymes glutamate synthase and glutamine synthase increased in activity from day 0 to 21 for both SF and NSF tissues. Enzyme activities for glutamate dehydrogenase were similar in both treatments to day 10 in culture but subsequently diverged, with activities in NSF cultures being substantially greater than those of SF cultures by day 21. Taken together, labeling and enzyme data indicate that nitrogen metabolism is enhanced during culture, especially in SF tissues at the time of promeristemoid formation, and in non‐organ‐forming tissue senescence‐like metabolism was exhibited later in culture.

Activities of Ribulose Bisphosphate Carboxylase and Phosphoenolpyruvate Carboxylase and 14C-Bicarbonate Fixation during in Vitro Culture of Pinus radiata Cotyledons

The activities of ribulose bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC), as indicators of autotrophic and nonautotrophic CO(2) fixation, were measured in excised cotyledons of Pinus radiata D. Don cultured for 21 days under shoot-forming (SF) and nonshoot-forming (NSF) conditions. The activity of RuBPC was found to increase in both SF and NSF cultures during the initial 5 days of culture. However, it leveled off from day 5 to day 10 and subsequently began to decrease until the end of the culture period under the SF conditions. In contrast, in the NSF cultures, RuBPC activity increased until day 15, when it was twofold higher than the maximum activity found in the SF cultures. An increase in PEPC activity of about 2.5 times the level of activity in freshly excised cotyledons was observed during the initial 5 days of culture under the SF conditions. PEPC activity began to decline after day 5 until it reached the level of activity seen in NSF cotyledons by day 15. In contrast, the activity of PEPC did not show any significant increase during the initial 10 days of culture under the NSF conditions. The K(m) (phosphoenolpyruvate) of PEPC from SF cotyledons was about 35% higher than that of NSF cotyledons. Cotyledons from two culture periods (days 5 and 15) were incubated for 15 seconds with NaH(14)CO(3). The label in the malate and asparatate fractions as a percentage of total (14)C incorporation was 3 times higher in the SF cotyledons than in the NSF cotyledons. A higher incorporation of (14)C into products of photosynthesis under the NSF conditions was also observed.

Initiation and propagation of Glycine max L. Merr.: Plants from tissue-cultured epicotyls

A successful technique for the initiation and proliferation of shoots from epicotyl tissue of soybean, Glycine max, has been developed and is described. Fertile plants were recovered. Seeds were germinated on Murashige and Skoog medium containing 5 μM benzyladenine. Explanted epicotyl sections were induced to form callus and shoots on Schenk and Hildebrandt medium containing 5.2 mM monobasic ammonium phosphate, 74 μM 3-aminopyridine, and 20 μM kinetin for five weeks. Shoot proliferation was maintained on N6 medium containing 1.75 mM ammonium sulfate, 2.1 nM picloram, and 0.1 μM benzyladenine. Shoots rooted on Gamborg's B5 medium without growth regulators. Shoot-forming cultures were maintained for 60 months. Although all varieties tested produced shoots, some variation in numbers of shoots obtained was observed.

Biochemical markers of embryogenesis in tissue cultures of the maize inbred B73

Stable embryogenic, organogenic and undifferentiated cell lines of the maize ( Zea mays L.) inbred B73 were used to assess the value of using isozyme analyses and the composition of secreted polysaccharides to identify embryogenic cells. Esterase, glutamate dehydrogenase, alcohol dehydrogenase and β-glucosidase all possessed developmentally regulated isozymes but only esterase and glutamate dehydrogenase could be used to distinguish between embryogenic and shoot-forming cultures. Embryogenic callus and suspension cultures secreted a musilagenous polysaccharide whose production was stimulated by 2,4-dichlorophenoxyacetic acid (2,4-D). This polysaccharide was different from root slime and corn hull gum and may be related to the ‘cementing layer’ in maize kernels.

Digitoxin Biosynthesis in Isolated Mesophyll Cells and Cultured Cells of <italic>Digitalis</italic>

In the laminae of Digitalis, most of the digitoxin present is found in the mesophyll. A new method for determining the amount of digitoxin biosynthesis using a digitoxin antibody was devised to estimate this activity in isolated mesophyll cells and cultured cells. Isolated mesophyll cells showed significant activity, which suggests that the site of biosynthesis and the accumulation of cardenolides in a lamina of Digitalis is mainly in the mesophyll cells. Of five liquid cultures of D. purpurea; green shoot-forming cultures, white shoot-forming cultures, root-forming cultures, undifferentiated green cells and undifferentiated white cells, the green shoot-forming cultures had the highest activity. The white shoot-forming cultures had about one-third the activity of the green shoot-forming cultures, and the other three cultures had very low activity. No stimulatory effect of light was found during the 48-h incubation.

Photoautotrophic Culture of Undifferentiated Cells and Shoot-Forming Cultures of <italic>Digitalis purpurea</italic> L.

Undifferentiated cells and shoot-forming cultures of Digitalis purpurea L. were grown photoautotrophically under 1% CO2. During 3 weeks of culture, the undifferentiated cells multiplied 3-fold and the shoot-forming cultures 2-fold on a fresh weight basis. The chlorophyll content, ribulose 1,5-bisphosphate carboxylase activity, Hill reaction activity of the isolated chloroplasts and photosynthetic O2 evolution of the photoautotrophically grown cultures were somewhat higher than the values of the corresponding photomixotrophic cultures. The digitoxin contents, however, were not improved by photoautotrophic culture.

Studies on the production of Digitalis cardenolides by plant tissue culture, Part V. Jar fermenter culture of shoot-forming cultures of Digitalis purpurea L. using a revised medium.

A revised medium for digitoxin production by shoot-forming cultures of Digitalis purpurea L. was established. Using this medium, jar fermenter culture of the shoot-forming cultures was performed. Approximately 25 g fresh weight of cells was inoculated into a 3-liter jar fermenter containing 2.5 liter of the medium, and cultured for 15 days at 28°C with aeration of 1.4 vvm without mechanical agitation. The jar was illuminated continuously throughout the culture period. The cell yield was 38.5 g dry weight per jar and the total digitoxin formation was 1610μg per jar. The optimum rate of aeration was 1.4 vvm and mechanical agitation had little effect.

Studies on the production of digitalis cardenolides by plant tissue culture. Part IV. Effects of mineral salts, initial pH and precursors on digitoxin formation by shoot-forming cultures of Digitalis purpurea L. grown in liquid media.

Shoot-forming cultures of Digitalis purpurea L. were grown in various modifications of Murashige-Skoog medium. The effects of CaCl2, MgSO4, FeSO4 coupled with Na2-EDTA, minor elements, initial pH of the medium, some organic solvents as precursor solvents, and precursors on their growth and digitoxin formation were investigated. Reduction of the basal CaCl2 concentration by two-thirds promoted both digitoxin formation and shoot-differentiation while a threefold increase repressed these. Ca++ proved to be responsible for this phenomenon. A threefold increase in the MgSO4 concentration improved digitoxin formation, Mg++ being responsible for this improvement. The optimal concentration of FeSO4 coupled with Na2-EDTA was similar to that in the MS basal medium. A threefold increase in the concentration of either KI, Na2MoO4 or CuSO4 improved digitoxin formation, the optimal pH of the medium before autoclaving being 6.Among methanol, ethanol, propylene glycol, dimethyl sulfoxide and acetone as the precursor solvents, methanol and acetone were relatively the least inhibitive at 2%. Progesterone at 0.1mg/100ml medium improved the digitoxin content per flask to 180% of that of the control cultures, whereas no promotive effect from cholesterol was observed.

Studies on the Production of Digitalis Cardenolides by Plant Tissue Culture III. Effects of Nutrients on Digitoxin Formation by Shoot-Forming Cultures of Digitalis purpurea L. Grown in Liquid Media

Shoot-forming cultures of Digitalis purpurea L. were grown in various modifications of Murashige-Skoog medium. The effects of vitamins, natural extracts, plant growth substances, carbon and nitrogen sources and phosphate on their growth and digitoxin formation were investigated. Sucrose, glucose and raffinose were suitable carbon sources for both growth and digitoxin formation. The optimum concentration of sucrose was 3%. Reduction of the basal nitrogen concentration by one-third increased the digitoxin content per unit weight without suppression of growth. The optimal ratio of nitrate-nitrogen to ammonium-nitrogen was 2, as provided in the basal medium. A threefold increase in the phosphate concentration improved both growth and digitoxin content. Thiamine.HCl and myo-inositol were required for digitoxin formation, whereas glycine, nicotinic acid and pyridoxine.HCl, usual components of the medium, were not required for either growth or digitoxin formation during the three passages examined. Coconut milk improved growth with no reduction in the digitoxin content per unit weight. Gibberellic and abscisic acids at 0.01 to 0.1 mg per liter slightly improved digitoxin formation. Kinetin had no clear positive effect on either growth or digitoxin formation.

Studies on the Production of Digitalis Cardenolides by Plant Tissue Culture

Undifferentiated, highly chlorophyllous cell cultures; undifferentiated white cell cultures; green, shoot-forming cultures; and white, shoot-forming cultures of Digitalis purpurea L. were established and subcultured every 3 weeks in liquid media in the light or in the dark. The digitoxin content, the chlorophyll content, and the ribulose bisphosphate carboxylase activity of these cultures were assayed. The light-grown, green, shoot-forming cultures accumulated considerable amounts of digitoxin (about 20 to 40 micrograms per gram dry weight), and the white, shoot-forming cultures without chloroplasts accumulated about one-third that amount of digitoxin. The chlorophyll content and the ribulose bisphosphate carboxylase activity of the undifferentiated green cells were about the same as they were in the green, shoot-forming cultures, but the digitoxin content of the former was extremely low (about 0.05 to 0.2 microgram per gram dry weight), which is about the same as that in undifferentiated white cells without chloroplasts. Thus, it was concluded that the chloroplasts are not essential for the synthesis of digitoxin in Digitalis cells. The optimum concentrations of the tested compounds for accumulation of digitoxin were: benzyladenine, 0.01 to 1 milligram per liter; indoleacetic acid, 0.1 to 1 milligram per liter; alpha-naphthaleneacetic acid; 0.1 milligram per liter; and 2,4-dichlorophenoxyacetic acid, 0.01 milligram per liter.